BackgroundThe high crystallinity of cellulosic biomass myofibrils as well as the complexity of their intermolecular structure is a significant impediment for biofuel production. Cloning of celB-, celC-encoded cellulases (cel12B and cel8C) and peh-encoded polygalacturonase (peh28) from Pectobacterium carotovorum subsp. carotovorum (Pcc) was carried out in our previous study using Escherichia coli as a host vector. The current study partially characterizes the enzymes’ molecular structures as well as their catalytic performance on different substrates which can be used to improve their potential for lignocellulosic biomass conversion.Resultsβ-Jelly roll topology, (α/α)6 antiparallel helices and right-handed β-helices were the folds identified for cel12B, cel8C, and peh28, respectively, in their corresponding protein model structures. Purifications of 17.4-, 6.2-, and 6.0-fold, compared to crude extract, were achieved for cel12B and cel8C, and peh28, respectively, using specific membrane ultrafiltrations and size-exclusion chromatography. Avicel and carboxymethyl cellulose (CMC) were substrates for cel12B, whereas for cel8C catalytic activity was only shown on CMC. The enzymes displayed significant synergy on CMC but not on Avicel when tested for 3 h at 45 °C. No observed β-glucosidase activities were identified for cel8C and cel12B when tested on p-nitrophenyl-β-d-glucopyranoside. Activity stimulation of 130% was observed when a recombinant β-glucosidase from Pcc was added to cel8C and cel12B as tested for 3 h at 45 °C. Optimum temperature and pH of 45 °C and 5.4, respectively, were identified for all three enzymes using various substrates. Catalytic efficiencies (k cat/K m) were calculated for cel12B and cel8C on CMC as 0.141 and 2.45 ml/mg/s respectively, at 45 °C and pH 5.0 and for peh28 on polygalacturonic acid as 4.87 ml/mg/s, at 40 °C and pH 5.0. Glucose and cellobiose were the end-products identified for cel8C, cel12B, and β-glucosidase acting together on Avicel or CMC, while galacturonic acid and other minor co-products were identified for peh28 action on pectin.ConclusionsThis study provides some insight into which parameters should be optimized when application of cel8C, cel12B, and peh28 to biomass conversion is the goal.

Background: Biomass produced as a byproduct from the β-mannanase production process by Aspergillus tamarii NRC 3was evaluated as a biosorbent for the removal and recovery of some heavy metal ions.Results: Under optimal conditions, the isolated strain recorded the highest β-mannanase activity (31.88 Uml −1 ). Thus, the biomass produced from mannanase production process as a byproduct was evaluated as a biosorbent for the removal and recovery of some heavy metal ions from aqueous solutions and an industrial wastewater. The fungal biomass was found to be efficient for the removal of Cu +2 and some heavy metal ions. The biosorption process of copper(II) by Aspergillus tamarii NRC 3 biomass was affected by changing of time, temperature, pH, metal ions concentration, the presence of some heavy metals, and biomass concentration. The rate of Cu +2 uptake from Cu +2 solution proceeded rapidly, and it appeared to be virtually complete during the initial 5 min (92%); the maximum uptake of Cu +2 appeared at 30°C, pH 5, and biomass concentration 5 g w/w. On the other hand, the fungal biomass was to remove considerable proportion of Pb 2+ , Co +2 , Ni 2+ , Fe +3 , and Cr 3+ in addition to Cu 2+ . The uptake of Cu +2 by pretreated biomass was studied. Recovery of the sorbed metal ions by desorbing agents and the potential reuse of the regenerated biomass in metal ions uptake (reloading) were evaluated. Conclusions: Aspergillus tamarii NRC 3 biomass seems to be quite feasible in the removal of heavy metal ions especially Cu +2 from aqueous solutions.

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Summary Lignocellulosic biomass conversion using cellulases/polygalacturonases is a process that can be progressively influenced by several determinants involved in cellulose microfibril degradation. The current paper focuses on the kinetics and thermodynamics of thermal inactivation of recombinant E. coli cellulases, cel12B, cel8C, and a polygalacturonase, peh 28, derived from Pectobacterium carotovorum sub sp. carotovorum. Several consensus motifs conferring the enzymes’ thermal stability in both cel12B and peh28 model structures have been detailed earlier, which were confirmed for the three enzymes through the current study of their thermal inactivation profiles over the 20-80 °C range using the respective activities on carboxymethylcellulose and polygalacturonic acid. Kinetic constants and half-lives of thermal inactivation, inactivation energy, plus inactivation entropies, enthalpies and Gibbs free energies, revealed high stability, less conformational change and protein unfolding for cel12B and peh28 due to thermal denaturation compared to cel8C. The apparent thermal stability of peh28 and cel12B, along with their hydrolytic efficiency on a lignocellulosic biomass conversion as reported previously, makes these enzymes candidates for various industrial applications. Analysis of the Gibbs free energy values suggests that the thermal stabilities of cel12B and peh28 are entropy-controlled over the tested temperature range.