Endoglucanase effects on energy consumption in the mechanical fibrillation of cellulose fibers into nanocelluloses.

Enzymatic processing is considered a promising approach for advancing environmentally friendly industrial processes, such as the use of endoglucanase (EG) enzyme in the production of nanocellulose. However, there is ongoing debate regarding the specific properties that make EG pretreatment effective in isolating fibrillated cellulose. To address this issue, we investigated EGs from four glycosyl hydrolase (GH) families (5, 6, 7, and 12) and examined the roles of the three-dimensional structure and catalytic features, with a focus on the presence of a carbohydrate binding module (CBM). Using eucalyptus Kraft wood fibers, we produced cellulose nanofibrils (CNFs) through mild enzymatic pretreatment, followed by disc ultra-refining. Comparing the results with the control (without pretreatment), we observed that GH5 and GH12 enzymes (without CBM) reduced fibrillation energy by approximately 15 %. The most significant energy reduction, 25 and 32 %, was achieved with GH5 and GH6 linked to CBM, respectively. Notably, these CBM-linked EGs improved the rheological properties of CNF suspensions without releasing soluble products. In contrast, GH7-CBM exhibited significant hydrolytic activity, resulting in the release of soluble products, but did not contribute to a reduction in fibrillation energy. This discrepancy can be attributed to the large molecular weight and wide cleft of GH7-CBM, which led to the release of soluble sugars but had little impact on fibrillation. Our findings suggest that the improved fibrillation observed with EG pretreatment is primarily driven by efficient enzyme adsorption on the substrate and modification of the surface viscoelasticity (amorphogenesis), rather than hydrolytic activity or release of products.

Kinetic changes in cellulose properties during defibrillation into microfibrillated cellulose and cellulose nanofibrils by ultra-refining.

Defibrillation of cellulose fibers can lead to the isolation of microfibrillated cellulose (MFC) or cellulose nanofibrils (CNF) with intrinsic properties suitable for various applications. However, to what extent these properties are preserved, enhanced, gained or lowered during defibrillation and how they are related remains unclear. In this study, a kinetic study of the ultra-refining of bleached eucalyptus Kraft pulp (BEKP) in a disc ultra-refiner was performed and characterized in terms of physical-structural, morphological and thermal properties and their interactions and compromises. Defibrillation of BEKP to MFC substantially decreased the fiber diameter and increased viscosity, surface area and morphological heterogeneity. It also led to a remarkable increase in transparency and essentially did not alter the thermostability but significantly degraded the crystallinity. A higher degree of defibrillation to isolate CNF led to fibers with smaller diameter and increased diameter uniformity but required a substantial amount of energy to only marginally increase viscosity and transparency. Crystallinity and thermostability were not altered, comparing with CMF. In conclusion, most changes occurred during the defibrillation of BEKP to CMF. Further defibrillation to CNFs with smaller diameters and better uniformity did not significantly reflect on other important structural cellulose physical properties, despite the much higher energy consumption and degree of defibrillation.

Cloning, heterologous expression and biochemical characterization of a non-specific endoglucanase family 12 from Aspergillus terreus NIH2624.

The cellulases from Glycoside Hydrolyses family 12 (GH12) play an important role in cellulose degradation and plant cell wall deconstruction being widely used in a number of bioindustrial processes. Aiming to contribute toward better comprehension of these class of the enzymes, here we describe a high-yield secretion of a endoglucanase GH12 from Aspegillus terreus (AtGH12), which was cloned and expressed in Aspergillus nidulans strain A773. The purified protein was used for complete biochemical and functional characterization. The optimal temperature and pH of the enzyme were 55°C and 5.0 respectively, which has high activity against ß-glucan and xyloglucan and also is active toward glucomannan and CMC. The enzyme retained activity up to 60°C. AtGH12 is strongly inhibited by Cu2+, Fe2+, Cd2+, Mn2+, Ca2+, Zn2+ and EDTA, whereas K+, Tween, Cs+, DMSO, Triton X-100 and Mg2+ enhanced the enzyme activity. Furthermore, SAXS data reveal that the enzyme has a globular shape and CD analysis demonstrated a prevalence of a ß-strand structure corroborating with typical ß-sheets fold commonly found for other endoglucanases from GH12 family.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Aspergillus terreus endo-ß-1,4-glucanase from glycoside hydrolase family 12.

Endoglucanases are important enzymes that are involved in the modification and degradation of cellulose. Filamentous fungi such as Aspergillus terreus are effective biomass degraders in nature owing to their capacity to produce an enzymatic arsenal of glycoside hydrolases, including endoglucanase from glycoside hydrolase family 12 (GH12). The A. terreus GH12 endoglucanase was cloned and overexpressed in A. nidulans, purified and crystallized. A single crystal was obtained from a solution consisting of 2 M ammonium sulfate, 5%(v/v) 2-propanol. X-ray diffraction data were collected to a resolution of 1.85 Å using synchrotron radiation and a preliminary molecular-replacement solution was obtained in the trigonal space group P3(2)21. The unit-cell parameters were a = b = 103.24, c = 48.96 Å.