(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 > Streptosporangiales: NE > Nocardiopsaceae: NE > Marinactinospora: NE > Marinactinospora thermotolerans: NE > Marinactinospora thermotolerans DSM 45154: 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 MLTHSISPEETDGGRRTPLSRMSRLAARVGVTLSLAAGLTAGVTAPAHAS NPYERGPAPTESSVTAVRGYFDTDTDTVSSLVSGFGGGTIYYPTDTSEGT FGGVVIAPGYTASQSSMAWMGHRIASQGFVVFTIDTITRYDQPDSRGRQI EAALDYLVEDSDVADRVDGNRLAVMGHSMGGGGTLAAAENRPELRAAIPL TPWHLQKNWSDVEVPTMIIGAENDTVASVRTHSIPFYESLDEDLERAYLE LDGASHFAPNISNTVIAKYSISWLKRFVDEDERYEQFLCPPPDTGLFSDF SDYRDSCPHTT
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: Catalytic Features and Thermal Adaptation Mechanisms of a Deep Sea Bacterial Cutinase-Type Poly(Ethylene Terephthalate) Hydrolase Liu Y, Liu C, Liu H, Zeng Q, Tian X, Long L, Yang J Ref: Front Bioeng Biotechnol, 10:865787, 2022 : PubMed
Poly (ethylene terephthalate) (PET) plastic is chemically inert and persistent. Massive quantities of PET waste end up in landfill sites and oceans, posing major global pollution concerns. PET degrading enzymes with high efficiency provide plastic recycling and bioremediation possibilities. Here, we report a novel cutinase, MtCut with distinct catalytic behaviors, derived from the deep sea Nocardiopsaceae family strain. Biochemical analyses showed MtCut efficiently hydrolyzed PET at ambient temperatures and in an exo-type manner. The activity and stability of MtCut were enhanced by the addition of calcium ions. Notably, no hydrolysis products inhibition was observed during PET depolymerization, suggesting MtCut is a better biocatalyst when compared to other PET hydrolases. In addition, structural components associated with thermal adaptation were investigated using molecular dynamic (MD) simulations, and key regions regulating MtCut thermostability were identified. Our biochemical and structural analyses of MtCut deepen the understanding of PET hydrolysis by cutinases, and provide invaluable insights on improvement and performance engineering strategies for PET-degrading biocatalysts.
Marinactinospora thermotolerans DSM 45154 Cutinase PET hydrolase
