Characterisation of two thermostable glycosyl hydrolases from the extremely acidophilic archaeon Cuniculiplasma divulgatum S5

Abstract

Enzymes from extremophilic microorganisms can withstand harsh industrial conditions, such as extreme pH and temperatures, making them superior biocatalysts compared to mesophilic enzymes. Cuniculiplasma divulgatum is a novel, extremely acidophilic archaeon discovered originally in acidic waters from copper mine sites in North Wales, UK, and Spain. These sites contain mostly hyperacidophilic, mesophilic and potentially thermo- and psychrotolerant microorganisms.

The C. divulgatum S5 genome was sequenced in 2016, revealing the presence of 22 genes encoding putative glycosyl hydrolases (GH), including three GH1 family enzymes. GH1 is a group of glycosyl hydrolase enzymes present in prokaryotes and eukaryotes, which have a broad substrate range and catalyze the hydrolysis of glycosidic bonds in various carbohydrates. GH1 enzymes play a pivotal role in the conversion of lignocellulosic biomass into reducing sugars, which are essential for the production of ethanol and other valuable products. Two GH1 enzymes from C. divulgatum, CIB12 and CIB13 (sharing 54% amino acid sequence identity), were earlier successfully cloned and expressed. The research presented in this thesis describes the results of biochemical characterization and structural analysis of CIB12 and CIB13.

In this project, both CIB12 and CIB13 were purified and biochemically characterized using both chromogenic and natural substrates for glycosyl hydrolases (GH). Screening of purified proteins against two panels of 23 chromogenic and 20 natural GH substrates revealed the highest hydrolytic activity against pNP-β-D-glucopyranoside and cellobiose in both enzymes. Both enzymes were most active at pH 6-7 and 50-60 °C, with CIB12 activity sharply reducing at 65 °C and higher temperatures, while CIB13 surprisingly retained significant activity (30-50 % of maximal) at 80 °C and even 90 °C. Both enzymes were activated by NaCl, with maximal activity observed at 0.25 M NaCl for CIB12 (up to a two-fold increase) and 3.5 M NaCl for CIB13 (a three-fold increase). The crystal structures of both enzymes were determined earlier (PDB codes 8U7F and 8U7G) and revealed a classical (α/β)8 TIM barrel fold with the active site located inside the barrel close to the C-terminal ends of β-strands.Both crystal structures show protein dimers with two tightly associated monomers containing two small molecule ligands bound close to two catalytic glutamate residues, Glu204 and Glu388 in CIB12 and Glu204 and Glu385 in CIB13. Structure-based alanine replacement mutagenesis confirmed the critical role of catalytic Glu residues and identified additional active site residues important for activity.

Phylogenetic analysis of CIB12 and CIB13 placed them in the GH1 family of enzymes, showing close evolutionary relationships with glycosidases from various archaeal and bacterial species. Comparative sequence analysis highlighted conserved catalytic residues and provided insights into their potential substrate preferences.Thus, in this study, two novel GH1 glycosyl hydrolases from the extremely acidophilic archaeon C. divulgatum were recombinantly expressed, purified, and biochemically characterized. With natural substrates, both enzymes were active against cellobiose and lactose with a preference for cellobiose, and therefore they were classified as cellobiases (EC 3.2.1.21). Both enzymes exhibit significant tolerance to high temperatures and have enhanced activities at higher salt concentrations.

Biochemical, structural characterization, and phylogenetic analysis of CIB12 and CIB13 provide a solid base for future enzyme optimization using protein engineering, thereby contributing to the development of a suite of extremophilic carbohydrate-active enzymes for potential industrial applications, particularly processes related to biomass conversion, biofuel production, and other applications in food industry involving the breakdown of lactose and cellobiose.

Date of Award	19 Jan 2024
Original language	English
Awarding Institution	Bangor University
Sponsors	Bangor University
Supervisor	Peter Golyshin (Supervisor) & Alexandre Iakounine (Supervisor)