Presence Of Cellulose-binding Domain Research Articles

504 “Chimeric sorting” as an effective tool for selection of genetically modified dermal fibroblasts to be used in fundamental or applied skin research

A technology has been developed for efficient isolation of a stable pool of HIF1a overexpressing mouse fibroblasts using a high-throughput selection of cell clones on a cellulose carrier. The pHIF1a-CBD-PDGFR genetic construct has been generated containing a EYFP reporter, a target sequence of HIF1a gene (a key factor of adaptation to hypoxia in wound healing and regeneration) and a CBD/PDGFR chimeric gene construct. The presence of CBD (cellulose-binding domain) / PDGFR (platelet-derived growth factor receptor) chimeric construct in protein-producing cells enables their specific binding to cellulose carrier thus providing a possibility for cell selection based on their adhesive phenotype (“chimeric sorting”). For genetic modification, a line of immortalized postnatal mouse dermal fibroblasts was used (dp3T3cell line). This cell line has been developed using 3T3 protocol and combines the basic features of primary dermal fibroblasts with the experimental advantages of immortal cells. The high-throughput selection of HIF1a overexpressing cells was performed using BD FACSAria™ Fusion Cell Sorter (with EYFP as a reporter) or by chimeric sorting on day 5th after dp3T3 cell transfection (electroporation) using half of transfected cells for each approach (0.9±0.2x10ˆ6). The CBD-PDGFR driven chimeric sorting was performed on cellulose filter papers, 75g/cm2. In 3 weeks chimeric sorting allowed to obtain 3±1x10ˆ6 stably transfected cells with changed morphology (less stellate and more elongate), while FACS sorting yielded 8 colonies with 0.6±0.3x10ˆ5 cells each with only 3 colonies exhibiting modified phenotype. Thus, “chimeric sorting” of transfected fibroblasts on a cellulose carrier appeared to be more effective in selection of HIF1a overexpressing cells as compared to conventional FACS. The advantageous efficiency of this approach was reflected in both its specificity (low level of contamination with cells of original phenotype) and its productivity (higher yield of stably transfected cells).

Purification and characterization of two endoglucanases from Melanocarpus sp. MTCC 3922

This study reports the purification and characterization of endoglucanases (EG I and EG II) from a newly isolated thermophilic fungus, Melanocarpus sp. MTCC 3922. The molecular weight of EG I and EG II as with SDS-PAGE and p I were ∼40 and 50 kDa, and ∼4.0 and 3.6, respectively. EG I and EG II were optimally active at 50 and 70 °C, and pH 6.0 and 5.0, respectively. EG I was active over a broad range of pH (5.0–7.0), whereas, loss of activity was observed as the temperature was increased from 50 to 80 °C. However, EG II was active over pH 4.0–6.0 and temperature 40–80 °C. The presence of mercaptoethanol and SDS inhibited the EG I activity but showed no negative effect on EG II. Both the endoglucanases showed higher activity against barley-β-glucan as compared to CMC. K m values of EG I and EG II for barley-β-glucan were lower than CMC. Turn over number ( K cat) and catalytic efficiency ( K cat/ K m) values of both the endoglucanases were higher with barley-β-glucan as substrate than CMC. EG I showed affinity for Avicel indicating the presence of cellulose binding domains (CBD) whereas, EG II was found to lack CBD.

The enhancement of the cellulolytic activity of cellobiohydrolase I and endoglucanase by the addition of cellulose binding domains derived from Trichoderma reesei

The effect of isolated cellulose binding domains (CBDs) on the hydrolysis of filter paper and microcrystalline cellulose by both cellobiohydrolase I and endoglucanase, was studied. CBDs were obtained by proteolysis from cellulases using a scaled-up variant of our previous method. Experiments were performed for different enzyme/substrate ratios in both the absence and presence of CBDs. Hydrolysis of filter paper by intact cellobiohydrolase I in the presence of additional CBDs was found to have a synergistic effect, leading to an increase of the sugar production of up to 30%. The effect was less pronounced using microcrystalline cellulose, where an increase up to 16% was observed. Similar trends were found during the hydrolysis of both substrates by endoglucanase.

Effects of agitation level on the adsorption, desorption, and activities on cotton fabrics of full length and core domains of EGV (Humicola insolens) and CenA (Cellulomonas fimi).

The activities (at pH 7 and 50 degrees C) of purified EGV (Humicola insolens) and CenA (Cellulomonas fimi) were determined on cotton fabrics at high and low levels of mechanical agitation. Similar activity measurements were also made by using the core domains of these cellulases. Activity experiments suggested that the presence of cellulose binding domains (CBDs) is not essential for cellulase performance in the textile processes, where high levels of mechanical agitation are applied. The binding reversibilities of these cellulases and their cores were studied by dilution of the treatment liquor after equilibrium adsorption. EGV showed low percentage of adsorption under both levels of agitation. It was observed that the adsorption/desorption processes of cellulases are enhanced by higher mechanical agitation levels and that the binding of cellulase with CBD of family I (EGV) is more reversible than that of CBD of the cellulase of family II (CenA).

Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp

The action of monocomponent Trichoderma reesei endoglucanases (EG I, EG II; EC 3.2.1.4) and cellobiohydrolases (CBH I, CBH II; EC 3.2.1.91) and their core proteins was compared using isolated celluloses and bleached chemical pulp. The presence of cellulose binding domain (CBD) in the intact enzymes did not affect their action against soluble substrates. In the case of insoluble isolated celluloses and the chemical pulp the presence of CBD enhanced the enzymatic hydrolysis of cellulose. The effect of CBD was more pronounced in the cellobiohydrolases, hydrolysing mainly crystalline cellulose, than in the endoglucanases which were more efficient in hydrolysing amorphous cellulose. The pulp properties measured, that is, viscosity and strength after PFI refining, were equally affected by the treatment with intact enzymes and corresponding core proteins, suggesting that the presence of CBD in intact cellulases affects mainly the cellulose hydrolysis level and less the mode of action of T. reesei cellulases in pulp. The better beatability of the bleached chemical pulp treated with intact endoglucanases than that treated with the corresponding core proteins suggests that the presence of CBD in endoglucanases could, however, result in beneficial effects on pulp properties.