Vasileva, D. | bioRxiv Science

bioRxiv2024

Identification and characterization of substrate- and product-selective nylon hydrolases

Enzymes have evolved to rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but only a limited group of related enzymes have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 diverse enzymes from the N-terminal nucleophile hydrolase superfamily with 30-50% pairwise amino acid identity. We found that nearly 40% of the enzymes had substantial nylon hydrolase activity, in many cases comparable to that of the best-characterized nylon hydrolase, NylC. There was no relationship between phylogeny and activity, nor any evidence of prior selection for nylon hydrolase activity. Several newly-identified hydrolases showed significant substrate selectivity, generating up to 20-fold higher product titers with Nylon 6,6 versus Nylon 6. Finally, we determined the crystal structure and oligomerization state of a Nylon 6,6-selective hydrolase to elucidate structural factors that could affect activity and selectivity. These new enzymes provide insights into the widespread potential for nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases. SignificanceNylons are common industrial polyamides with few recycling options. As an alternative to mechanical or chemical recycling, enzymes may provide a selective and energy-efficient route to deconstruct nylons from mixed waste. Several nylon hydrolases have been identified, most notably NylC, but these enzymes are all closely related and demonstrated similar activity and substrate range. In this work, we investigated a diverse set of enzymes and showed that nylon hydrolase activity is common, providing new insights into the evolution of microbial nylon hydrolysis. Unlike NylC, several enzymes demonstrated unprecedented substrate selectivity, preferentially hydrolyzing Nylon 6,6 compared to Nylon 6. These enzymes can be used to understand substrate selectivity in nylon hydrolysis and to engineer enzymes for nylon recycling from mixed waste.

biochemistry↗

bioRxiv2020

Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Computational analyses suggested that this bias is due to the action of restriction-modification systems whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that the intensity of recombination is uniform across the genome without concentration into hotspots. Unexpectedly, our results revealed that large species-level genetic distance did not affect key recombination parameters. Our study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.

microbiology↗