MALDI-TOF MS analysis of cellodextrins and xylo-oligosaccharides produced by hindgut homogenates of Reticulitermes santonensis.

Hindgut homogenates of the termite Reticulitermes santonensis were incubated with carboxymethyl cellulose (CMC), crystalline celluloses or xylan substrates. Hydrolysates were analyzed with matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS). The method was first set up using acid hydrolysis analysis to characterize non-enzymatic profiles. Commercial enzymes of Trichoderma reesei or T. longibrachiatum were also tested to validate the enzymatic hydrolysis analysis. For CMC hydrolysis, data processing and visual display were optimized to obtain comprehensive profiles and allow rapid comparison and evaluation of enzymatic selectivity, according to the number of substituents of each hydrolysis product. Oligosaccharides with degrees of polymerization (DPs) ranging from three to 12 were measured from CMC and the enzymatic selectivity was demonstrated. Neutral and acidic xylo-oligosaccharides with DPs ranging from three to 11 were measured from xylan substrate. These results are of interest for lignocellulose biomass valorization and demonstrated the potential of termites and their symbiotic microbiota as a source of interesting enzymes for oligosaccharides production.

Isolation of amylolytic, xylanolytic, and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere.

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO2 or CO2/H2) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacillus subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce ß-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed ß-glucosidase, amylase, cellulase, and xylanase activities. Finally, B. subtilis strain ABGx produced xylanase and amylase.

Identification and characterization of a new xylanase from Gram-positive bacteria isolated from termite gut (Reticulitermes santonensis).

Termites are world champions at digesting lignocellulosic compounds, thanks to cooperation between their own enzymes and exogenous enzymes from microorganisms. Prokaryotic cells are responsible for a large part of this lignocellulolytic activity. Bacterial enzyme activities have been demonstrated in the higher and the lower termite gut. From five clones of Gram-positive bacteria isolated and identified in a previous work, we constructed a genomic DNA library and performed functional screening for alpha-amylase, beta-glucosidase, and xylanase activities. One candidate, Xyl8B8, showed xylanase activity. Sequence analysis of the genomic insert revealed five complete ORFs on the cloned DNA (5746bp). Among the encoded proteins were a putative endo-1,4-beta-xylanase (XylB8) belonging to glycoside hydrolase family 11 (GH11). On the basis of sequence analyses, genomic DNA organization, and phylogenetic analysis, the insert was shown to come from an actinobacterium. The mature xylanase (mXylB8) was expressed in Escherichia coli and purified by affinity chromatography and detected by zymogram analysis after renaturing. It showed maximal xylanase activity in sodium acetate buffer, pH 5.0 at 55 °C. Its activity was increased by reducing agents and decreased by Cu(2+), some detergents, and chelating agents. Its substrate specificity appeared limited to xylan.

Effect of extraction pH on techno-functional properties of crude extracts from wild cardoon (Cynara cardunculus L.) flowers.

In the present paper, techno-functional properties of rennet derived from C. cardunculus, prepared at various pH (from 3 to 6), were investigated. It was found that the extract prepared at pH 3 had the best milk-clotting properties (MCA/PA ratio). It presented also the highest enzyme content, determined by densitometry analysis on 1D and 2D gels electrophoresis. Formation of milk gels produced by the extracts and chymosin was monitored using dynamic rheology and turbiscan. It can be assumed that by lowering rennet pH, milk pH decreases, causing a significant increase of curd firmness and both elastic (G') and viscous (G″) moduli. Results obtained by turbiscan showed similar ΔBS values of gels produced, after 2h, by chymosin and the crude extract at pH 3. In conclusion, C. cardunculus extract prepared at low pH has the potential to be employed as an efficient milk-clotting agent in the production of dairy products.

Identification of proteins from wild cardoon flowers (Cynara cardunculus L.) by a proteomic approach.

Proteomic approach was applied to identify total proteins, particularly the enzymatic content, from wild cardoon flowers. As the selection of an appropriate sample preparation method is the key for getting reliable results, two different extraction/precipitation methods (trichloroacetic acid and phenol/ammonium acetate) were tested on fresh and lyophilized flowers. After two-dimensional electrophoresis (2D-E) separations, a better protein pattern was obtained after phenol extraction from lyophilized flowers. Only 46 % of the total analyzed spots resulted in a protein identification by mass spectrometry MALDI-TOF. Four proteases (cardosins A, E, G, and H), which have become a subject of great interest in dairy technology, were identified. They presented molecular weights and isoelectric points very close and high levels of homology between matched peptides sequences. The absence of the other cardosins (B, C, D, and F) could be an advantage, as it reduces the excessive proteolytic activity that causes bitter flavors and texture defects, during cheese making.

Looking for phosphate-accumulating bacteria in activated sludge processes: a multidisciplinary approach.

Over the past decades, an increasing need in renewable resources has progressively appeared. This trend concerns not only fossil fuels but also mineral resources. Wastewater and sewage sludge contain significant concentrations in phosphate and can be considered as a fertilizer source of the utmost importance. In wastewater treatment plants, the biological uptake of phosphate is performed by a specific microbiota: the phosphate-accumulating organisms. These microorganisms are recovered in sewage sludge. Here, we aimed to investigate the occurrence of phosphate accumulators in four wastewater treatment plants. A 16S metagenetic analysis identified the main bacterial phyla extracted from the aerobic treatment: α-Proteobacteria, ß-Proteobacteria, and Sphingobacteria. An enrichment stage was performed to stimulate the specific growth of phosphate-accumulating bacteria in an acetate medium. An analysis of metabolic activities of sulfur and phosphorus highlighted strong modifications related to phosphorus and much less distinguishable effects with sulfur. A solid acetate medium containing 5-Br-4-Cl-3-indolyl phosphate was used to select potential phosphate-accumulating bacteria from the enriched consortia. The positive strains have been found to belong in the genera Acinetobacter, Corynebacterium, and Pseudomonas. Finally, electron microscopy was applied to the strains and allowed to confirm the presence of polyphosphate granules. Some of these bacteria contained granules the size of which exceeded 100 nm.

Isolation and cultivation of xylanolytic and cellulolytic Sarocladium kiliense and Trichoderma virens from the gut of the termite Reticulitermes santonensis.

The purpose of this work was the isolation and cultivation of cellulolytic and xylanolytic microorganisms extracted from the gut of the lower termite Reticulitermes santonensis. Microcrystalline cellulose (with and without lignin) and beech wood xylan were used as diets instead of poplar wood in order to select cellulose and hemicellulose-degrading fungi. The strain Sarocladium kiliense (Acremonium kiliense) CTGxxyl was isolated from the termites fed on xylan, while the strain Trichoderma virens CTGxAviL was isolated from the termites fed on cellulose (with and without lignin). Both molds were cultivated in liquid media containing different substrates: agro-residues or purified polymers. S. kiliense produced maximal ß-glucosidase, endo-1,4-ß-D-glucanase, exo-1,4-ß-D-glucanase and endo-1,4-ß-D-xylanase activities of 0.103, 3.99, 0.53, and 40.8 IU/ml, respectively. T. virens produced maximal ß-xylosidase, endo-1,4-ß-D-glucanase, exo-1,4-ß-D-glucanase, and endo-1,4-ß-D-xylanase activities of 0.38, 1.48, 0.69, and 426 IU/ml. The cellulase and the xylanase of S. kiliense, less common than T. virens, were further investigated. The optimal activity of the xylanase was observed at pH 9-10 at 60 °C. The cellulase showed its maximal activity at pH 10, 70 °C. Zymography identified different xylanases produced by both molds, and some fragment sizes were highlighted: 35, 100, and 170 kDa for S. kiliense and 20, 40, 80, and 170 kDa for T. virens. In both cases, endo-1,4-ß-D-xylanase activities were confirmed through mass spectrometry.

Symbiont diversity in Reticulitermes santonensis (Isoptera: Rhinotermitidae): investigation strategy through proteomics.

The complex microbial community living in the hindgut of lower termites includes prokaryotes, flagellates, yeasts, and filamentous fungi. Many microorganisms are found in the termite gut, but only a few are thought to be involved in symbiotic association to participate in cellulose digestion. Proteomics provides analyses from both taxonomical and functional perspectives. We aimed to identify symbiont diversity in the gut of Reticulitermes santonensis (Feytaud), via complementary electrospray ionization associated to ion trap tandem mass spectrometry (LC-MS/MS) and two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry analysis. One specific challenge to the study of lower termites is the relatively few data available on abundant symbiotic flagellates. Analysis based on LC-MS/MS revealed few protein families showing assignments to eukaryotes and the taxonomic origin of highly represented actins could not be established. Tubulins proved to be the most suitable protein family with which to identify flagellate populations from hindgut samples using LC-MS/MS, compared with other protein families, although this method targeted few prokaryotes in our assay. Similarly, two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry did not succeed in identifying flagellate populations, but did permit the identification of most of the prokaryotic components of the symbiotic system. Finally, fungi and yeasts were identified by both methods. Owing to the lack of sequenced genes in flagellates, targeting tubulins for LC-MS/MS could allow fingerprints of flagellate populations to be established. Experimental and technical improvements might increase the efficiency of identification of prokaryotic populations in the near future, based on metaproteomic development.

Isolation and cultivation of a xylanolytic Bacillus subtilis extracted from the gut of the termite Reticulitermes santonensis.

The aim of this work was the isolation of xylanolytic microorganisms from the digestive tract of the termite Reticulitermes santonensis. The reducing sugars released after the hydrolysis of xylans can be further fermented to provide bioethanol. A xylanolytic strain of Bacillus subtilis was isolated from the hindgut of the termite and displayed amylase and xylanase activities. The bacterium was grown on media containing agricultural residues: wheat bran, wheat distiller's grains, and rapeseed oil cake. Wheat bran led to the highest induction of xylanase activity, although the development of the strain was less fast than in the other media. It was possible to reach maximal xylanase activities of 44.3, 33.5, and 29.1 I.U./ml in the media containing wheat bran, wheat distiller's grains, and rapeseed oil cake, respectively. Mass spectrometry identified a wide range of xylose oligomers, highlighting an endoxylanase activity. The enzyme was stable up to 45 °C and displayed an optimal pH close to 8.

New glucosidase activities identified by functional screening of a genomic DNA library from the gut microbiota of the termite Reticulitermes santonensis.

ß-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying ß-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose.