(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Proteobacteria: NE > Betaproteobacteria: NE > Burkholderiales: NE > Comamonadaceae: NE > Caldimonas: NE > Caldimonas manganoxidans: 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 MSPSTLSTILLAACAALAAPGLAQAQYQQGPDPTASALERNGPFAIRSTS VSRTSVSGFGGGRLYYPTASGTYGAIAVSPGFTGTSSTMTFWGERLASHG FVVLVIDTITLYDQPDSRARQLKAALDYLATQNGRSSSPIYRKVDTSRRA VAGHSMGGGGSLLAARDNPSYKAAIPMAPWNTSSTAFRTVSVPTMIFGCQ DDSIAPVSSHAIPFYNAIPNSTRKNYVEIRNDDHFCVMNGGGHDATLGKL GISWMKRFVDNDTRYSPFVCGAEYNRVVSSYEVSRSYNNCPY
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.
Title: New insights into the function and global distribution of polyethylene terephthalate (PET) degrading bacteria and enzymes in marine and terrestrial metagenomes Danso D, Schmeisser C, Chow J, Zimmermann W, Wei R, Leggewie C, Li X, Hazen T, Streit WR Ref: Applied Environmental Microbiology, 84:e2773, 2018 : PubMed
Polyethylene terephthalate (PET) is one of the most important synthetic polymers used nowadays. Unfortunately, the polymers accumulate in nature and until now, no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly alpha/beta-hydrolases like cutinases and related enzymes (E.C. 3.1.-). Currently, only a small number of such enzymes are well characterized. Within this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 GB of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the IMG data base detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. Thereby, two novel and thermostable enzymes with high potential for downstream application were in part characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phylum of Actinobacteria, Proteobacteria and Bacteroidetes Within the Proteobacteria, the Beta-, Delta- and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum of the Bacteroidetes appear to be the main host of PET hydrolase genes rather than Actinobacteria or Proteobacteria as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although few PET hydrolases are already known it is still unknown how frequent they appear and which main bacterial phyla they are affiliated with. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed are occurring at very low frequencies in the environment. Further it was possible to link them to phyla which were previously unknown to harbor such enzymes. This work contributes novel knowledge to the phylogenetic relationship, the recent evolution and the global distribution of PET hydrolases. Finally, we describe biochemical traits of four novel PET hydrolases.
