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Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture Philipp Knyphausen , Mariana Rangel Pereira , Paul Brear , Marko Hyvonen , Lutz Jermutus , Florian Hollfelder Nature Communications 14 DOI: 10.1038/s41467-023-36099-7 Diamond Proposal Number(s): [18548, 25402] Open Access Show Abstract ▼ Abstract: Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving protease catalysts is challenging and often labour intensive. Here, we describe a generic method to accelerate this process based on yeast display. We introduce the protease selection system A2Mcap that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2Mcap for protease engineering we exemplify the directed activity and specificity evolution of six serine proteases. This resulted in a variant of Staphylococcus aureus serin-protease-like (Spl) protease SplB, an enzyme used for recombinant protein processing, that no longer requires activation by N-terminal signal peptide removal. SCHEMA-based domain shuffling was used to map the specificity determining regions of Spl proteases, leading to a chimeric scaffold that supports specificity switching via subdomain exchange. The ability of A2Mcap to overcome key challenges en route to tailor-made proteases suggests easier access to such reagents in the future.	Feb 2023
I04-Macromolecular Crystallography	From data mining of Chitinophaga sp. genome to enzyme discovery of a hyperthermophilic metallocarboxypeptidase Gabriela Cabral Fernandes , Elwi Guillermo Machado Sierra , Paul Brear , Mariana Rangel Pereira , Eliana G. M. Lemos Microorganisms 9 DOI: 10.3390/microorganisms9020393 Diamond Proposal Number(s): [25402] Open Access Show Abstract ▼ Abstract: For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications.	Feb 2021
I02-Macromolecular Crystallography	From a metagenomic source to a high-resolution structure of a novel alkaline esterase Mariana Rangel Pereira , Thaís Carvalho Maester , Gustavo Fernando Mercaldi , Eliana Gertrudes De Macedo Lemos , Marko Hyvonen , Andrea Balan Applied Microbiology And Biotechnology 279 DOI: 10.1007/s00253-017-8226-4 Diamond Proposal Number(s): [14043] Show Abstract ▼ Abstract: Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of α/β hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-Ǻ resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met213 and Phe217) that block the substrate channel. A small increase in the reaction velocity for p-nitrophenyl butyrate and p-nitrophenyl valerate hydrolysis was observed. Activity against p-nitrophenyl acetate was reduced. The functional and structural characterization of Est8 is explored in comparison with orthologues.	Mar 2017

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