Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost.

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by ß-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.

Multiple fluorescence labeling and two dimensional FTIR-13C NMR heterospectral correlation spectroscopy to characterize extracellular polymeric substances in biofilms produced during composting.

Knowledge on the structure and function of extracellular polymeric substances (EPS) in biofilms is essential for understanding biodegradation processes. Herein, a novel method based on multiple fluorescence labeling and two-dimensional (2D) FTIR-(13)C NMR heterospectral correlation spectroscopy was developed to gain insight on the composition, architecture, and function of EPS in biofilms during composting. Compared to other environmental biofilms, biofilms in the thermophilic (>55 °C) and cooling (mature) stage of composting have distinct characteristics. The results of multiple fluorescence labeling demonstrated that biofilms were distributed in clusters during the thermophilic stage (day 14), and dead cells were detected. In the mature stage (day 26), the biofilm formed a continuous layer with a thickness of approximately 20-100 µm around the compost, and recolonization of cells at the surface of the compost was easily observed. Through 2D FTIR-(13)C NMR correlation heterospectral spectroscopy, the following trend in the ease of the degradation of organic compounds was observed: heteropolysaccharides > cellulose > amide I in proteins. And proteins and cellulose showed significantly more degradation than heteropolysaccharides. In summary, the combination of multiple fluorescence labeling and 2D correlation spectroscopy is a promising approach for the characterization of EPS in biofilms.

[Comparison of Extraction Methods of Extracellular Polymeric Substances from Activated Sludge].

Extracellular polymeric substances (EPSs) are biosynthetic polymers of microbial origin in the sludge activation process and crucially affect the properties of sludge in biological wastewater treatment reactors, such as the formation of sludge flocs, stabilization of sludge structure, and protection of microbes against noxious environmental conditions. However, the EPS extraction efficiency differs significantly according to the extraction method used. In this study, soluble EPSs and loosely bound EPSs can be extracted by centrifugation first and tightly bound EPSs in activated sludge require additional eight treatments for extraction, respectively. Three physical methods (centrifugation, sonication, and heating) and five chemical methods (cation exchange resin, NaOH, formaldehyde+NaOH, EDTA, and formaldehyde+EDTA) were tested, and the content and composition of TB-EPS were analyzed. Meanwhile, the functional groups and elements in TB-EPS were investigated. Results showed that the heating method did not introduce exogenous substances during the EPS extraction process and that the destruction of cells from this method was relatively slight. Heating was shown to be a gentle and efficient method in this study. The three-dimensional excitation and emission matrix (EEM) fluorescence spectroscopy showed that the cation exchange resin method had good extraction effect on humic-like and protein-like substances. As to fulvic-acid-like substances, NaOH was better than the other seven methods. Infrared spectroscopy showed that no notable difference appeared in the functional groups of the TB-EPS extracted by physical methods, whereas chemical methods induced big differences and showed particular bands that did not appear in the TB-EPS extracted by physical methods. Overall, the amounts of TB-EPS elements extracted using chemical methods were greater than those extracted using physical methods. In conclusion, a method must be selected and established for each case, taking into consideration the experimental purpose, and the most appropriate method should be chosen carefully.