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1.
Sci Rep ; 13(1): 1267, 2023 01 23.
Article in English | MEDLINE | ID: mdl-36690710

ABSTRACT

Agitation is a commonly encountered stress for enzymes during all stages of production and application, but investigations that aim to improve their tolerance using topological engineering have yet to be reported. Here, the plastic-degrading enzyme IsPETase was cyclized in a range of topologies including a cyclic monomer, cyclic dimer and catenane using SpyTag/SpyCatcher technologies, and their tolerance towards different stresses including mechanical agitation was investigated. The cyclic dimer and catenane topologies were less susceptible to agitation-induced inactivation resulting in enhancement of polyethylene terephthalate (PET) degradation. While contrary to conventional belief, cyclic topologies did not improve tolerance of IsPETase towards heat or proteolytic treatment, the close proximity of active sites in the dimeric and catenane variants was found to enhance PET conversion into small soluble products. Together, these findings illustrate that it is worthwhile to explore the topology engineering of enzymes used in heterogeneous catalysis as it improves factors that are often overlooked in homogeneous catalysis studies.


Subject(s)
Catenanes , Polyethylene Terephthalates , Polyethylene Terephthalates/chemistry , Plastics , Hydrolases/metabolism , Hot Temperature
2.
Org Biomol Chem ; 19(18): 3983-4001, 2021 05 12.
Article in English | MEDLINE | ID: mdl-33978044

ABSTRACT

The cyclisation of polypeptides can play a crucial role in exerting biological functions, maintaining stability under harsh conditions and conferring proteolytic resistance, as demonstrated both in nature and in the laboratory. To date, various approaches have been reported for polypeptide cyclisation. These approaches range from the direct linkage of N- and C- termini to the connection of amino acid side chains, which can be applied both in reaction vessels and in living systems. In this review, we categorise the cyclisation approaches into chemical methods (e.g. direct backbone cyclisation, native chemical ligation, aldehyde-based ligations, bioorthogonal reactions, disulphide formation), enzymatic methods (e.g. subtiligase variants, sortases, asparaginyl endopeptidases, transglutaminases, non-ribosomal peptide synthetases) and protein tags (e.g. inteins, engineered protein domains for isopeptide bond formation). The features of each approach and the considerations for selecting an appropriate method of cyclisation are discussed.


Subject(s)
Proteins
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