The use of phylogenetics and ancestral sequence reconstruction to identify improved halophilic enzymes for the biodegradation of poly(R-3-hydroxybutyrate)

Evolutionary analysis combined with ancestral sequence reconstruction (ASR) was utilized to calculate the taxonomic relationship between poly(hydroxybutyrate) depolymerase (PHBase) enzymes belonging to the halophilic genus Halomonas. Enzymes cluster into four distinct clades that differ in sequence length and composition. Like many of the previously studied PHBase proteins, there is a high degree of heterogeneity at the genus level. Ancestor sequences were calculated for each tree node using a maximum likelihood approach. The most ancestral sequence and one extant enzyme from each of the clades were expressed in Escherichia coli, purified to homogeneity, and characterized. The ancestral enzyme binds substrate more efficiently, is kinetically faster, and is more stable at elevated temperatures compared to the extant enzymes. Hence, an evolutionary phylogenetic approach is a viable alternative to other techniques for identifying optimized enzymes for industrial PHB degradation.