Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASL^tag). The anchored chimeric H⁵-SspCA was active for the CO₂ hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASL^tag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions. Graphical Abstract

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASL^tag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H⁵) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the β-glycoside hydrolase (SsβGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.

We present a new class of DNA‐based nanoswitches that, upon enzymatic repair, could undergo a conformational change mechanism leading to a change in fluorescent signal. Such folding‐upon‐repair DNA nanoswitches are synthetic DNA sequences containing O⁶‐methyl‐guanine (O⁶‐MeG) nucleobases and labelled with a fluorophore/quencher optical pair. The nanoswitches are rationally designed so that only upon enzymatic demethylation of the O⁶‐MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into an optically active triplex DNA structure. We have first characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and Molecular Dynamics simulations. We then demonstrated that the folding‐upon‐repair DNA nanoswitches are suitable and specific substrates for different methyltransferase enzymes including the human homologue (hMGMT) and they allow the screening of novel potential methyltransferase inhibitors.

The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O⁶-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.