Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9.

Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs.

Recombinant Prevotella melaninogenica α-1,3 glucanase and Capnocytophaga ochracea α-1,6 glucanase as enzymatic tools for in vitro degradation of S. mutans biofilms.

Dental biofilms represent a serious oral health problem playing a key role in the development of caries and other oral diseases. In the present work, we cloned and expressed in E. coli two glucanases, Prevotella melaninogenica mutanase (PmGH87) and Capnocytophaga ochracea dextranase (CoGH66), and characterized them biochemically and biophysically. Their three-dimensional structures were elucidated and discussed. Furthermore, we tested the capacity of the enzymes to hydrolyze mutan and dextran to prevent formation of Streptococcus mutans biofilms, as well as to degrade pre- formed biofilms in low and abundant sugar conditions. The percentage of residual biofilm was calculated for each treatment group in relation to the control, as well as the degree of synergism. Our results suggest that both PmGH87 and CoGH66 are capable of inhibiting biofilm formation grown under limited or abundant sucrose conditions. Degradation of pre-formed biofilms experiments reveal a time-dependent effect for the treatment with each enzyme alone. In addition, a synergistic and dose-dependent effects of the combined enzymatic treatment with the enzymes were observed. For instance, the highest biomass degradation was 95.5% after 30 min treatment for the biofilm grown in low sucrose concentration, and 93.8% after 2 h treatment for the biofilm grown in sugar abundant condition. Strong synergistic effects were observed, with calculated degree of synergism of 5.54 and 3.18, respectively and their structural basis was discussed. Jointly, these data can pave the ground for the development of biomedical applications of the enzymes for controlling growth and promoting degradation of established oral biofilms.

Staphylococcus aureus microbial biofilms degradation using cellobiose dehydrogenase from Thermothelomyces thermophilus M77.

This work reports biochemical characterization of Thermothelomyces thermophilus cellobiose dehydrogenase (TthCDHIIa) and its application as an antimicrobial and antibiofilm agent. We demonstrate that TthCDHIIa is thermostable in different ionic solutions and is capable of oxidizing multiple mono and oligosaccharide substrates and to continuously produce H2O2. Kinetics measurements depict the enzyme catalytic characteristics consistent with an Ascomycota class II CDH. Our structural analyses show that TthCDHIIa substrate binding pocket is spacious enough to accommodate larger cello and xylooligosaccharides. We also reveal that TthCDHIIa supplemented with cellobiose reduces the viability of S. aureus ATCC 25923 up to 32 % in a planktonic growth model and also inhibits its biofilm growth on 62.5 %. Furthermore, TthCDHIIa eradicates preformed S. aureus biofilms via H2O2 oxidative degradation of the biofilm matrix, making these bacteria considerably more susceptible to gentamicin and tetracycline.

Enzymatic production of xylooligosaccharides from corn cobs: Assessment of two different pretreatment strategies.

Corn cobs (CCs) are abundant xylan-rich agricultural wastes. Here, we compared CCs XOS yields obtained via two different pretreatment routs, alkali and hydrothermal, using a set of recombinant endo- and exo-acting enzymes from GH10 and GH11 families, which have different restrictions for xylan substitutions. Furthermore, impacts of the pretreatments on chemical composition and physical structure of the CCs samples were evaluated. We demonstrated that alkali pretreatment route rendered 59 mg of XOS per gram of initial biomass, while an overall XOS yield of 115 mg/g was achieved via hydrothermal pretreatment using a combination of GH10 and GH11 enzymes. These results hold a promise of ecologically sustainable enzymatic valorization of CCs via "green" and sustainable XOS production.