An insight into the thermostability of a pair of xylanases: the role of hydrogen bonds

Xylanases are enzymes that are very tolerant to temperature. Their potential use in several biotechnological applications, such as animal food manufacture and pulp bleaching, is due to their intrinsic thermostability. The present report deals with two xylanases, the mesophilic xylanase from Bacillus circulans, BCX, and the thermophilic xylanase from Thermomyces lanuginosus,TLX. These enzymes belong to family 11, and they are the most structurally similar mesophilic-thermophilic pair. Molecular dynamics simulations were employed to investigate the factors responsible for the different thermostabilities exhibited by these structurally similar enzymes. Their active site is their most rigid region, and it is equally rigid at all temperatures. Inter and intramolecular interactions, hydrogen bonds in particular, are the key to the main differences between BCX and TLX. The intramolecular hydrogen bonds and salt bridges are important for maintenance of the backbone rigidity even at high temperature, and the highly solvated surface is a clear optimization in TLX compared with BCX. The main differences between these two enzymes can be found on the fingers domain, which indicates that this domain must be the target for the site-directed mutagenesis responsible for improving the temperature tolerance of this family of enzymes.

Thermostability of heterophile antibodies from human sera infected with Schistosoma mansoni and geo-helminths: an immuno-metric statistical analysis

Antibody in human sera that induces lysis of sheep erythrocytes in hemolytic assay was investigated. The present study showed that the presence in serum of the thermostable cytolytic anti-sheep red blood cells antibodies is dependent on the Schistosoma mansoni infection, and this is more frequent in adults than in children. The thermostable characteristic of hemolysins in normal sera was not dependent on the presence of Ascaris lumbricoides, Trichuris trichiura or hookworm geo-helminths. Further, thermostable complement-activating heterophile antibodies were noticed in children in association with massive number of S. mansoni eggs. The results were obtained by using the z- and the chi-square tests. The z-test allows us to formulate a one-sided alternative, i.e., a tendency of one of the attributes. On the other hand, the chi-square test analyzes the independence between attributes by using a contingency table. Besides the obtained results being interesting in the field of schistosomiasis mansoni, they can provide a new insight into the use of statistics in medical science.

Contribution of electrostatic interactions, compactness and quaternary structure to protein thermostability: lessons from structural genomics of Thermotoga maritima.

Studies of the structural basis of protein thermostability have produced a confusing picture. Small sets of proteins have been analyzed from a variety of thermophilic species, suggesting different structural features as responsible for protein thermostability. Taking advantage of the recent advances in structural genomics, we have compiled a relatively large protein structure dataset, which was constructed very carefully and selectively; that is, the dataset contains only experimentally determined structures of proteins from one specific organism, the hyperthermophilic bacterium Thermotoga maritima, and those of close homologs from mesophilic bacteria. In contrast to the conclusions of previous studies, our analyses show that oligomerization order, hydrogen bonds, and secondary structure play minor roles in adaptation to hyperthermophily in bacteria. On the other hand, the data exhibit very significant increases in the density of salt-bridges and in compactness for proteins from T.maritima. The latter effect can be measured by contact order or solvent accessibility, and network analysis shows a specific increase in highly connected residues in this thermophile. These features account for changes in 96% of the protein pairs studied. Our results provide a clear picture of protein thermostability in one species, and a framework for future studies of thermal adaptation.

Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations

Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 angstrom resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis, for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting beta-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.

Characterization and comparison of thermostability of purified beta-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus

The thermophilic fungus Thermoascus aurantiacus 179-5 and the mesophilic Aureobasidium pullulans ER-16 were cultivated in corn-cob by solid state fermentation for P-glucosidase production. After fermentation both enzymes were purified. The beta-glucosidases produced by the strains A. pullulans and T aurantiacus were most active at pH 4.0-4.5 and 4.5, with apparent optimum temperatures at 80 and 75 degrees C, respectively. Surprisingly, the enzyme produced by the mesophilic A. pullulans was stable over a wider range of pH (4.5-9.5 against 4.5-6.5) and more thermostable (98% after 1 h at 75 degrees C against 98% after 1 h at 70 degrees C) than the enzyme from the thermophilic T. aurantiacus. The t((1/2)) at 80 degrees C were 90 and 30 min for A. pullulans and T. aurantiacus, respectively. beta-Glucosidase thermoinactivation followed first-order kinetics and the energies of denaturation were 414 and 537 kJ mol(-1) for T. aurantiacus and A. pullulans, respectively. The result showed that beta-glucosidase obtained from the mesophilic A. pullulans is more stable than that obtained from the thermophilic T. aurantiacus. (C) 2007 Elsevier Ltd. All rights reserved.

Improvement of Aspergillus niger glucoamylase thermostability by directed evolution

Directed evolution was used to improve the thermostability of Aspergillus niger glucoamylase (GA) expressed in Saccharomyces cerevisiae. A starch-plate assay developed to screen GA mutants for thermostability gave results consistent with those of irreversible thermoinactivation kinetic analysis. Several thermostable multiply-mutated GAs were isolated and characterized by DNA sequencing and kinetic analysis. Three new GA mutations, T62A, T290A and H391Y, have been identified that encode GAs that are more thermostable than wild-type GA, and that improve thermostability cumulatively. These individual mutations were combined with the previously constructed thermostable site-directed mutations D20C/A27C (forming a disulficle bond), S30P, and G137A to create a multiply-mutated GA designated THS8. THS8 GA is substantially more thermostable than wild-type GA at 8OoC, with a 5.1 kJ/mol increase in the free energy of therrnoinactivation, making it the most thermostable Aspergillus niger GA mutant characterized to date. THS8 GA and the singly-mutated GAs have specific activities and catalytic efficiencies (k(cat)/K-m) similar to those of wild-type GA.

THERMOSTABILITY OF XYLANOLYTIC ENZYMES PRODUCED BY LENTINULA EDODES UFV70

Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65 degrees C for 21 hours, losing only 15-25% activity. Fungus incubation at 50 degrees C for 12 hours and at 65 degrees C for 24 hours increased the amount of xylose produced.

Thermostability of xylanolytic enzymes produced by Lentinula edodes UFV70

Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65ºC for 21 hours, losing only 15-25% activity. Fungus incubation at 50ºC for 12 hours and at 65ºC for 24 hours increased the amount of xylose produced.

Enzyme engineering of aminotransferases for improved activity and thermostability

The production by biosynthesis of optically active amino acids and amines satisfies the pharmaceutical industry in its demand for chiral building blocks for the synthesis of various pharmaceuticals. Among several enzymatic methods that allow the synthesis of optically active aminoacids and amines, the use of minotransferase is a promising one due to its broad substrate specificity and no requirement for external cofactor regeneration. The synthesis of chiral compounds by aminotransferases can be done either by asymmetric synthesis starting from keto acids or ketones, and by kinetic resolution starting from racemic aminoacids or amines. The asymmetric synthesis of substituted (S)-aminotetralin, an active pharmaceutical ingredient (API), has shown to have two major factors that contribute to increasing the cost of production. These factors are the raw material cost of biocatalyst used to produce it and product loss during biocatalyst separation. To minimize the cost contribution of biocatalyst and to minimize the loss of product, two routes have been chosen in this research: 1. To engineer the aminotransferase biocatalyst to have greater specific activity, and 2. Improve the engineering of the process by immobilization of biocatalyst in calcium alginate and addition of cosolvents. An (S)-aminotransferase (Mutant CNB03-03) was immobilized, not as purified enzyme but as enzyme within spray dried cells, in calcium alginate beads and used to produce substituted (S)-aminotetralin at 50 °C and pH 7 in experiments where the immobilized biocatalyst was recycled. Initial rate of reaction for cycle 1 (6 hr duration) was determined to be 0.258 mM/min, for cycle 2 (20 hr duration) it decreased by ~50% compared to cycle 1, and for cycle 3 (20 hr duration) it decreased by ~90% compared to cycle 1 (immobilized preparation consisted of 50 mg of spray dried cells per gram of calcium alginate). Conversion to product for each cycle decreased as well, from 100% in cycle 1 (About 50 mM), 80% in cycle 2, and 30% after cycle 3. This mutant was determined to be deactivated at elevated temperatures during the reaction cycle and was not stable enough to allow multiple cycles in its immobilized form. A new mutant aminotransferase was isolated by applying error-prone polymerase chain reaction (PCR) on the gene coding for this enzyme and screening/selection: CNB04-01. This mutant showed a significant improvement in thermostability in comparison to CNB03-03. The new mutant was immobilized and tested under similar reaction conditions. Initial rate remained fairly constant (0.2 mM/min) over four cycles (each cycle with a duration of about 20 hours) with the mutant retaining almost 80% of initial rate in the fourth cycle. The final product concentrations after each cycle did not decrease during recycle experiments. Thermostability of CNB04-01 was much improved compared to CNB03-03. Under the same reaction conditions as stated above, the addition of co-solvents was studied in order to increase substituted tetralone solubility. Toluene and sodium dodecylsulfate (SDS) were used. SDS at 0.01% (w/v) allowed four recycles of the immobilized spray dried cells of CNB04-01, always reaching higher product concentration (80-85 mM) than the system with toluene at 3% (v/v) -70 mM-. The long term activity of immobilized CNB04-01 in a system with SDS 0.01% (w/v) at 50 °C, pH 7 was retained for three cycles (20 to 24 hours each one), reaching always final product concentration between 80-85 mM, but dropping precipitously in the fourth cycle to a final product concentration of 50 mM. Although significant improvement of immobilization on productivity and stability were observed using CNB04-01, another observation demonstrated the limitations of an immobilization strategy on reducing process costs. After analyzing the results of this experiment it was seen that a sudden drop occurred on final product concentration after the third recycle. This was due to product accumulation inside the immobilized preparation. In order to improve the economics of the process, research was focused on developing a free enzyme with an even higher activity, thus reducing raw material cost as well as improving biomass separation. A new enzyme was obtained (CNB05-01) using error-prone PCR and screening using as a template the gene derived from the previous improved enzyme. This mutant was determined to have 1.6 times the initial rate of CNB04-01 and had a higher temperature optimum (55°). This new enzyme would allow reducing enzyme loading in the reaction by five-fold compared to CNB03-03, when using it at concentration of one gram of spray dried cells per liter (completing the reaction after 20-24 hours). Also this mutant would allow reducing process time to 7-8 hours when used at a concentration of 5 grams of spray dried cells per liter compared to 24 hours for CNB03-03, assuming that the observations shown before are scalable. It could be possible to improve the economics of the process by either reducing enzyme concentration or reducing process time, since the production cost of the desired product is primarily a function of both enzyme concentration and process time.

Protein thermostability above 100°C: A key role for ionic interactions

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100°C. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100°C and 88°C, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104°C. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104°C over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.

Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.

Thermostability profile of Newcastle disease virus (strain I-2) following serial passages without heat selection

I-2 is an avirulent strain of Newcastle disease virus. During establishment of the I-2 strain master vaccine seed, a series of selection procedures was carried out at 56 degrees C in order to enhance heat resistance. This master seed is used to produce a working seed, which is then employed to produce the vaccine. These two passages are done without further heat selection; however, it is not known how rapidly and to what extent thermostable variants would be lost during further passage. The study was therefore conducted to determine the effect of passage on thermostability of strain I-2. The virus was serially passaged and at various passage levels samples were subjected to heat treatment at 56 degrees C for 120 min. The inactivation rates for infectivity and haemagglutinin (HA) titres were assayed by use of chicken embryonated eggs and HA test, respectively. Thermostability of HA and infectivity of I-2 virus were reduced after 10 and 5 passages, respectively, without heat selection at 56 degrees C. These results suggest that 5 more passages could be carried out between the working seed and vaccine levels without excessive loss of thermostability. This would result in increased vaccine production from a single batch of a working seed.

Singlet molecular oxygen trapping by the fluorescent probe diethyl-3,3 '-(9,10-anthracenediyl)bisacrylate synthesized by the Heck reaction

Singlet molecular oxygen O(2)((1)Delta(g)) is a potent oxidant that can react with different biomolecules, including DNA, lipids and proteins. Many polycyclic aromatic hydrocarbons have been studied as O(2)((1)Delta(g)) chemical traps. Nevertheless, a suitable modification in the polycyclic aromatic ring must be made to increase the yield of O(2)((1)Delta(g)) chemical trapping. With this goal, an anthracene derivative, diethyl-3,3 '-(9,10-anthracenediyl)bisacrylate (DADB), was obtained from the reaction of 9,10-dibromoanthracene and ethyl acrylate through the Heck coupling reaction. The coupling of ethyl acrylate with the anthracene ring produced a new lipophilic, esterified, fluorescent probe reactive toward O(2)((1)Delta(g)). This compound reacts with O(2)((1)Delta(g)) at a rate of k(r) = 1.69 x 10(6) M(-1) s(-1) forming a stable endoperoxide (DADBO(2)), which was characterized by UV-Vis, fluorescence, HPLC/MS and (1)H and (13)C NMR techniques. The photophysical, photochemical and thermostability features of DADB were also evaluated. Furthermore, this compound has the potential for great application in biological systems because it is easily synthetized in large amount and generates specific endoperoxide (DADBO(2)), which can be easily detected by HPLC tandem mass spectrometry (HPLC/MS/MS).

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.