Determination of fungal activity in modified wood by means of micro-calorimetry and determination of total esterase activity

Beech and pine wood blocks were treated with 1,3-dimethylol-4,5-dihydroxyethylen urea (DMDHEU) to increasing weight percent gains (WPG). The resistance of the treated specimens against Trametes versicolor and Coniophora puteana, determined as mass loss, increased with increasing WPG of DMDHEU. Metabolic activity of the fungi in the wood blocks was assessed as total esterase activity (TEA) based on the hydrolysis of fluorescein diacetate and as heat or energy production determined by isothermal micro-calorimetry. Both methods revealed that the fungal activity was related with the WPG and the mass loss caused by the fungi. Still, fungal activity was detected even in wood blocks of the highest WPG and showed that the treatment was not toxic to the fungi. Energy production showed a higher consistency with the mass loss after decay than TEA; higher mass loss was more stringently reflected by higher heat production rate. Heat production did not proceed linearly, possibly due to the inhibition of fungal activity by an excess of carbon dioxide

Perspectives on the use of transcriptomics to advance biofuels

As a field within the energy research sector, bioenergy is continuously expanding. Although much has been achieved and the yields of both ethanol and butanol have been improved, many avenues of research to further increase these yields still remain. This review covers current research related with transcriptomics and the application of this high-throughput analytical tool to engineer both microbes and plants with the penultimate goal being better biofuel production and yields. The initial focus is given to the responses of fermentative microbes during the fermentative production of acids, such as butyric acid, and solvents, including ethanol and butanol. As plants offer the greatest natural renewable source of fermentable sugars within the form of lignocellulose, the second focus area is the transcriptional responses of microbes when exposed to plant hydrolysates and lignin-related compounds. This is of particular importance as the acid/base hydrolysis methods commonly employed to make the plant-based cellulose available for enzymatic hydrolysis to sugars also generates significant amounts of lignin-derivatives that are inhibitory to fermentative bacteria and microbes. The article then transitions to transcriptional analyses of lignin-degrading organisms, such as Phanerochaete chrysosporium, as an alternative to acid/base hydrolysis. The final portion of this article will discuss recent transcriptome analyses of plants and, in particular, the genes involved in lignin production. The rationale behind these studies is to eventually reduce the lignin content present within these plants and, consequently, the amount of inhibitors generated during the acid/base hydrolysis of the lignocelluloses. All four of these topics represent key areas where transcriptomic research is currently being conducted to identify microbial genes and their responses to products and inhibitors as well as those related with lignin degradation/formation.clos

CelG from Clostridium cellulolyticum: a multidomain endoglucanase acting efficiently on crystalline cellulose.

The gene coding for CelG, a family 9 cellulase from Clostridium cellulolyticum, was cloned and overexpressed in Escherichia coli. Four different forms of the protein were genetically engineered, purified, and studied: CelGL (the entire form of CelG), CelGcat1 (the catalytic domain of CelG alone), CelGcat2 (CelGcat1 plus 91 amino acids at the beginning of the cellulose binding domain [CBD]), and GST-CBD(CelG) (the CBD of CelG fused to glutathione S-transferase). The biochemical properties of CelG were compared with those of CelA, an endoglucanase from C. cellulolyticum which was previously studied. CelG, like CelA, was found to have an endo cutting mode of activity on carboxymethyl cellulose (CMC) but exhibited greater activity on crystalline substrates (bacterial microcrystalline cellulose and Avicel) than CelA. As observed with CelA, the presence of the nonhydrolytic miniscaffolding protein (miniCipC1) enhanced the activity of CelG on phosphoric acid swollen cellulose (PASC), but to a lesser extent. The absence of the CBD led to the complete inactivation of the enzyme. The abilities of CelG and GST-CBD(CelG) to bind various substrates were also studied. Although the entire enzyme is able to bind to crystalline cellulose at a limited number of sites, the chimeric protein GST-CBD(CelG) does not bind to either of the tested substrates (Avicel and PASC). The lack of independence between the two domains and the weak binding to cellulose suggest that this CBD-like domain may play a special role and be either directly or indirectly involved in the catalytic reaction

Longitudinal melanonychia in children: a clinical and histopathologic study of 40 cases.

Very little has been published on longitudinal melanonychia in children. Our objective was to determine the nature of melanocytic lesions in pediatric patients with longitudinal or total melanonychia and to look for correlations between clinical and histologic features.Journal Articleinfo:eu-repo/semantics/publishe

Interaction between the endoglucanase CelA and the scaffolding protein CipC of the Clostridium cellulolyticum cellulosome.

The 5' end of the cipC gene, coding for the N-terminal part of CipC, the scaffolding protein of Clostridium cellulolyticum ATCC 35319, was cloned and sequenced. It encodes a 586-amino-acid peptide, including several domains: a cellulose-binding domain, a hydrophilic domain, and two hydrophobic domains (cohesin domains). Sequence alignments showed that the N terminus of CipC and CbpA of C. cellulovorans ATCC 35296 have the same organization. The mini-CipC polypeptide, containing a cellulose-binding domain, hydrophilic domain 1, and cohesin domain 1, was overexpressed in Escherichia coli and purified. The interaction between endoglucanase CelA, with (CelA2) and without (CelA3) the characteristic clostridial C-terminal domain called the duplicated-segment or dockerin domain, and the mini-CipC polypeptide was monitored by two different methods: the interaction Western blotting (immunoblotting) method and binding assays with biotin-labeled protein. Among the various forms of CelA (CelA2, CelA3, and an intermediary form containing only part of the duplicated segment), only CelA2 was found to interact with cohesin domain 1 of CipC. The apparent equilibrium dissociation constant of the CelA2-mini-CipC complex was 7 x 10(-9)M, which indicates that there exists a high affinity between these two proteins

Production of Heterologous and Chimeric Scaffoldins by Clostridium acetobutylicum ATCC 824

Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional

Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum.

The cellulolytic complex was isolated from Clostridium cellulolyticum grown on cellulose. Upon gel filtration, the complex was found to consist mainly of 600-kDa units, along with a 16-MDa aggregate. Its ability to degrade various substrates and its capacity to bind to the crystalline cellulose were measured. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and blotting analysis showed that all of the known cellulases of this organism are present in this complex. Three major components were observed: the first component, a noncatalytic, large (160-kDa) protein, was identified based on its ability to bind to the dockerin-containing cellulases as scaffolding protein CipC. The other two components, which had molecular masses of 94 and 80.6 kDa, were identified as CelE and CelF, respectively. The identified cellulases and some other components of the cellulosome were able to bind to a miniCipC1 construct. In addition to providing an extensive description of the system, the results of the present study confirm that the dockerin-cohesin domain interaction plays an essential role in the constitution of the cellulosome