Correlation of Structure, Function and Protein Dynamics in GH7 Cellobiohydrolases from \u3cem\u3eTrichoderma atroviride\u3c/em\u3e, \u3cem\u3eT. reesei\u3c/em\u3e and \u3cem\u3eT. harzianum\u3c/em\u3e

Background: The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. Results: The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. Conclusions: We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering

Static Analysis of Underground Ant Nest Structures

Ants are known as nature’s master builders, constructing a wide range of nests, from simple nests with a single channel linking several chambers to ‘underground palaces’ that are several metres wide. However, there is a lack of understanding of the mechanical performance of underground ant nest structures. In this paper, two underground ant nest structures are obtained and digitally modelled by means of liquid paraffin wax infusion shaping, manual excavation and industrial CT scanning, and a finite element model of the underground ant nest structure is established by means of numerical simulation. By simulating people of different weights standing above the nests, the stress distribution characteristics of each chamber in the nests under different pressures are obtained, and the mechanical properties of the underground ant nest structures under static loads are investigated

Static Analysis of Underground Ant Nest Structures

Ants are known as nature’s master builders, constructing a wide range of nests, from simple nests with a single channel linking several chambers to ‘underground palaces’ that are several metres wide. However, there is a lack of understanding of the mechanical performance of underground ant nest structures. In this paper, two underground ant nest structures are obtained and digitally modelled by means of liquid paraffin wax infusion shaping, manual excavation and industrial CT scanning, and a finite element model of the underground ant nest structure is established by means of numerical simulation. By simulating people of different weights standing above the nests, the stress distribution characteristics of each chamber in the nests under different pressures are obtained, and the mechanical properties of the underground ant nest structures under static loads are investigated

Whole-Cell PVA Cryogel-Immobilized Microbial Consortium LE-C1 for Xanthan Depolymerization

Xanthan is an extracellular heteropolysaccharide produced by the bacteria Xanthomonas campestris. Due to its unique properties, the polysaccharide and its derivatives are widely used in many industries, from food to biomedicine and oil production, that demands an efficient xanthan depolymerization method to adapt this polysaccharide for various applications. Unlike the known chemical approaches, biological methods are considered to be more environmentally friendly and less energy intensive. In laboratory conditions, we have isolated a bacterial community capable of reducing the xanthan viscosity. Identification of the individual isolates in the microbial community and their testing resulted in the consortium LE-C1, consisting of two microorganisms Paenibacillus phytohabitans KG5 and Cellulosimicrobium cellulans KG3. The specific activities of the overall xanthanase and auxiliary enzymes that may be involved in the xanthan depolymerization were as follows: xanthanase, 19.6 ± 0.6 U/g; β-glucosidase, 3.4 ± 0.1 U/g; α-mannosidase, 68.0 ± 2.0 U/g; β-mannosidase, 0.40 ± 0.01 U/g; endo-glucanase, 4.0 ± 0.1 U/g; and xanthan lyase, 2.20 ± 0.07 U/mg. In order to increase the efficiency of xanthan biodegradation, the LE-C1 whole cells were immobilized in a poly(vinyl alcohol) cryogel. The resulting regenerative biocatalyst was able to complete xanthan depolymerization within 40 cycles without loss of activity or degradation of the matrix

Antibacterial Properties of Fucoidans from the Brown Algae <i>Fucus vesiculosus</i> L. of the Barents Sea

Fucoidans, sulfated polysaccharides found in cell walls of brown algae, are considered as a promising antimicrobial component for various applications in medicine and the food industry. In this study, we compare the antibacterial properties of two fractions of fucoidan from the brown algae Fucus vesiculosus gathered in the littoral of the Barents Sea and sampled at different stages of purification. The crude fraction of fucoidan was isolated from algae by extraction with aqueous ethanol and sonication. The purified fraction was obtained by additional treatment of the crude fraction with a solution of calcium chloride. The structural features of both fractions were characterized in detail and their antibacterial effects against several Gram-positive and Gram-negative bacteria were compared by photometry, acridine orange staining assay, and atomic force microscopy. Fucoidan inhibited growth in all of the above microorganisms, showing a bacteriostatic effect with minimum inhibitory concentrations (MIC) in the range between 4 and 6 mg/mL, with E. coli being the most sensitive to both fractions. Changes in the chemical composition after treatment of the crude fraction with a solution of calcium chloride led to a decrease in the content of sulfates and uronic acids and diminished antibacterial activity

Biochemical and genetic characterization of a novel enzyme of pentitol metabolism: D-arabitol-phosphate dehydrogenase.

An enzyme with a specificity that has not been described previously, D-arabitol-phosphate dehydrogenase (APDH), has been purified from cell lysate of Enterococcus avium. SDS/PAGE indicated that the enzyme had a molecular mass of 41+/-2 kDa, whereas a molecular mass of 160+/-5 kDa was observed under non-denaturing conditions, implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have a narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into xylulose 5-phosphate and ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD(+) and NADP(+) were accepted as cofactors. Based on the partial protein sequences, the APDH gene was cloned. Homology comparisons place APDH within the medium-range dehydrogenase family. Unlike most members of this family, APDH requires Mn(2+) but no Zn(2+) for enzymic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system. Both biochemical evidence and protein sequence homology comparisons indicate that similar enzymes are widespread among the Gram-positive bacteria. Their apparent biological role is to participate in arabitol catabolism via the 'arabitol phosphate route', similar to the ribitol and xylitol catabolic routes described previously

α-Galactobiosyl units: thermodynamics and kinetics of their formation by transglycosylations catalysed by the GH36 α-galactosidase from Thermotoga maritima

Broad regioselectivity of α-galactosidase from Thermotoga maritima (TmGal36A) is a limiting factor for application of the enzyme in the directed synthesis of oligogalactosides. However, this property can be used as a convenient tool in studies of thermodynamics of a glycosidic bond. Here, a novel approach to energy difference estimation is suggested. Both transglycosylation and hydrolysis of three types of galactosidic linkages were investigated using total kinetics of formation and hydrolysis of pNP-galactobiosides catalysed by monomeric glycoside hydrolase family 36 α-galactosidase from T. maritima, a retaining exo-acting glycoside hydrolase. We have estimated transition state free energy differences between the 1,2- and 1,3-linkage (ΔΔG‡0 values were equal 5.34 ± 0.85 kJ/mol) and between 1,6-linkage and 1,3-linkage (ΔΔG‡0 = 1.46 ± 0.23 kJ/mol) in pNP-galactobiosides over the course of the reaction catalysed by TmGal36A. Using the free energy difference for formation and hydrolysis of glycosidic linkages (ΔΔG‡F − ΔΔG‡H), we found that the 1,2-linkage was 2.93 ± 0.47 kJ/mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44 ± 0.71 kJ/mol lower

The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides

In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly

Biochemical characterization of Aspergillus awamori exoinulinase: substrate binding characteristics and regioselectivity of hydrolysis

1H-NMR analysis was applied to investigate the hydrolytic activity of Aspergillus awamori inulinase. The obtained NMR signals and deduced metabolite pattern revealed that the enzyme cleaves off only fructose from inulin and does not possess transglycosylating activity. Kinetics for the enzyme hydrolysis of inulooligosaccharides with different degree of polymerization (d.p.) were recorded. The enzyme hydrolyzed both beta2,1- as well as beta2,6-fructosyl linkages in fructooligosaccharides. From the k(cat)/K(m) ratios obtained with inulooligosaccharides with d.p. from 2 to 7, we deduce that the catalytic site of the inulinase contains at least five fructosyl-binding sites and can be classified as exo-acting enzyme. Product analysis of inulopentaose and inulohexaose hydrolysis by the Aspergillus inulinase provided no evidence for a possible multiple-attack mode of action, suggesting that the enzyme acts exclusively as an exoinulinase