RESUMEN
The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.
Asunto(s)
Burkholderiales , Burkholderiales/enzimología , Burkholderiales/genética , Burkholderiales/metabolismo , Ácidos Ftálicos/metabolismo , Ácidos Ftálicos/química , Hidrolasas/metabolismo , Hidrolasas/genética , Hidrolasas/química , Solubilidad , Tereftalatos Polietilenos/metabolismo , Tereftalatos Polietilenos/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Modelos MolecularesRESUMEN
Parasites, such as the malaria parasite P. falciparum, are critically dependent on host nutrients. Interference with nutrient uptake can lead to parasite death and, therefore, serve as a successful treatment strategy. P. falciparum parasites cannot synthesise cholesterol, and instead source this lipid from the host. Here, we tested whether cholesterol uptake pathways could be 'hijacked' for optimal drug delivery to the intracellular parasite. We found that fluorescent cholesterol analogues were delivered from the extracellular environment to the intracellular parasite. We investigated the uptake and inhibitory effects of conjugate compounds, where proven antimalarial drugs (primaquine and artesunate) were attached to steroids that mimic the structure of cholesterol. These conjugated antimalarial drugs improved the inhibitory effects against multiple parasite lifecycle stages, multiple parasite species, and drug-resistant parasites, whilst also lowering the toxicity to human host cells. Steroids with introduced peroxides also displayed antimalarial activity. These results provide a proof-of-concept that cholesterol mimics can be developed as a drug delivery system against apicomplexan parasites with the potential to improve drug efficacy, increase therapeutic index, and defeat drug resistance.