(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Terrabacteria group: NE > Actinobacteria [phylum]: NE > Actinobacteria [class]: NE > Micromonosporales: NE > Micromonosporaceae: NE > Allorhizocola: NE > Allorhizocola rhizosphaerae: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MAMQLNKTQASPVAGHASRVWTRAIAAASLGILTAIAVPGPAMAADNPYQ RGPDPTRDSVAASRGTFATASTTVGSGNGFGAGFIYYPTDTSQGTFGAVA IVPGYTATWAAEGAWMGHWLASFGFVVIGIDTINRNDWDTARGTQLLAAL DYLTQRSTVRDRVDASRLAVMGHSMGGGGAMYAALQRPSLKAAVGLAPFS PSQNLNGMRVPTMLLAGQHDTTTTPASITSLYNGIPAATEKAYLELSGAG HGFPTSNNSVMMRKVIPWLKIFVDSDVRYTQFLCPLMDNTGIRSYQSTCP LLPGTPTPPNRYEAETSPAVCTGTIASNHTGYSGTGFCDGNNATNAYAQF TVNASAAGSMTLRVRFANGTTTARPASLIVNGSTVQTPSFEGTGAWTTWA TKTLTVTLNAGNNTIRFNPTTANGLPNLDYIEIAAP
Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 degreesC. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.
