Bacterial cellulose nanocrystals obtained through enzymatic and acidic routes: A comparative study of their main properties and in vitro biological responses.

Cellulose nanocrystals (CNCs) are crystalline domains isolated from cellulosic fibers. They have been utilized in a wide range of applications, such as reinforcing fillers, antibacterial agents and manufacturing of biosensors. Whitin this context, the aim of this work was to obtain and analyze CNCs extracted from bacterial nanocellulose (BNC) using two distinct methods combined with milling pre-treatment: an acidic hydrolysis using 64 % sulfuric acid and an enzymatic hydrolysis using a commercial cellulase enzyme mixture. The CNCs obtained from the enzymatic route (e-CNCs) were observed to be spherical nanoparticles with diameter of 56 ± 11 nm. In contrast, the CNCs from the acid hydrolysis (a-CNCs) appeared as needle-shaped nanoparticles with a high aspect ratio with lengths/widths of 158 ± 64 nm/11 ± 2 nm. The surface zeta potential (ZP) of the a-CNCs was -30,8 mV, whereas the e-CNCs has a potential of +2.70 ± 3.32 mV, indicating that a-CNCs consisted of negatively charged particles with higher stability in solution. Although the acidic route resulted in nanocrystals with a slightly higher crystallinity index compared to the enzymatic route, e-CNCs was found to be more thermally stable than BNC and a-CNCs. Here, we also confirmed the safety of a-CNCs and e-CNCs using L929 cell line. Lastly, this article describes two different CNCs synthesis approaches that leads to the formation of nanoparticles with different dimensions, morphology and unique physicochemical properties. To the best of our knowledge, this is the first study to yield spherical nanoparticles as a result of BNC enzymatic treatment.

Functional characterization and comparative analysis of two heterologous endoglucanases from diverging subfamilies of glycosyl hydrolase family 45.

Lignocellulosic materials are abundant, renewable and are emerging as valuable substrates for many industrial applications such as the production of second-generation biofuels, green chemicals and pharmaceuticals. However, the recalcitrance and the complexity of cell wall polysaccharides require multiple enzymes for their complete conversion to oligo- and monosaccharides. The endoglucanases from GH45 family are a small and relatively poorly studied group of enzymes with potential industrial application. The present study reports cloning, heterologous expression and functional characterization of two GH45 endoglucanases from mesophilic fungi Gloeophyllum trabeum (GtGH45) and thermophilic fungi Myceliophthora thermophila (MtGH45), which belong to subfamilies GH45C and GH45A, respectively. Both enzymes have optimal pH 5.0 and melting temperatures (Tm) of 66.0 °C and 80.9 °C, respectively, as estimated from circular dichroism experiments. The recombinant proteins also exhibited different mode of action when incubated with oligosaccharides ranging from cellotriose to cellohexaose, generating mainly cellobiose and cellotriose (MtGH45) or glucose and cellobiose (GtGH45). The MtGH45 did not show activity against oligosaccharides smaller than cellopentaose while the enzyme GtGH45 was able to depolymerize cellotriose, however with lower efficiency when compared to larger oligosaccharides. Furthermore, both GHs45 were stable up to 70 °C for 24 h and useful to enhance initial glucan hydrolysis rates during saccharification of sugarcane pith by a mixture of cellulolytic enzymes. Recombinant GHs45 from diverging subfamilies stand out for differences in substrate specificity appearing as new tools for preparation of enzyme cocktails used in cellulose hydrolysis.