Unravelling biochemical and structural features of Bacillus licheniformis GH5 mannanase using site-directed mutagenesis and high-resolution protein crystallography studies.

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.

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.