Improving Trichoderma reesei Cel7B thermostability by targeting the weak spots.

For proteins that denature irreversibly, the denaturation is typically triggered by a partial unfolding, followed by a permanent change (e.g., aggregation). The regions that initiate the partial unfolding are named "weak spots". In this work, a molecular dynamics (MD) simulation and data analysis protocol is developed to identify the weak spots of Trichoderma reesei Cel7B, an important endoglucanase in cellulose hydrolysis, through assigning the local melting temperature (Tmp) to individual residue pairs. To test the predicted weak spots, a total of eight disulfide bonds were designed in these regions and all enhanced the enzyme thermostability. The increased stability, quantified by ΔT50 (which is the T50 difference between the mutant and the wild type enzyme), is negatively correlated with the MD-predicted Tmp, demonstrating the effectiveness of the protocol and highlighting the importance of the weak spots. Strengthening interactions in these regions proves to be a useful strategy in improving the thermostability of Tr. Cel7B.

Engineering a more thermostable blue light photo receptor Bacillus subtilis YtvA LOV domain by a computer aided rational design method.

The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the T m of the flavin mononucleotide based fluorescent protein Bacillus Subtilis YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.

The protein amide ¹H(N) chemical shift temperature coefficient reflects thermal expansion of the N-H···O=C hydrogen bond.

The protein amide (1)H(N) chemical shift temperature coefficient can be determined with high accuracy by recording spectra at different temperatures, but the physical mechanism responsible for this temperature dependence is not well understood. In this work, we find that this coefficient strongly correlates with the temperature coefficient of the through-hydrogen-bond coupling, (3h)J(NC'), based on NMR measurements of protein GB3. Parallel tempering molecular dynamics simulation suggests that the hydrogen bond distance variation at different temperatures/replicas is largely responsible for the (1)H(N) chemical shift temperature dependence, from which an empirical equation is proposed to predict the hydrogen bond thermal expansion coefficient, revealing responses of individual hydrogen bonds to temperature changes. Different expansion patterns have been observed for various networks formed by ß strands.

Combined effect of heavy metals and polycyclic aromatic hydrocarbons on urease activity in soil.

Actions and interactions between heavy metals (HM)-cadmium, zinc, lead, and polycyclic aromatic hydrocarbons (PAHs)-phenanthrene, fluoranthene, benzo(a)pyrene toward soil urease activity were studied after 7, 14, 21, and 28 days of exposure under controlled conditions. The experimental approach was based on the uniform design. Ten different contamination conditions were studied simultaneously, with 10 concentration levels for each pollutant. Data treatment was essentially based on the multiple regression technique. The results showed that Zn interacted more easily with PAHs than Pb or Cd. On the first 7 days of incubation, zinc alone reduced the urease activity more significantly than any other pollutants and no significant interactions between PAH and HM were observed. From 14 to 21 days of incubation, the interaction between Zn and benzo(a)pyrene decreased the soil urease activity. At 14 days, the interaction between Zn and phenanthrene was antagonistic (less than additive), while at 21 days it was synergistic (more than additive). At 28 days, the interaction between phenanthrene and fluoranthene was synergistic. This study indicated that the combined effect of PAH and HM on soil urease activity depends largely on the incubation time. Uniform design appears to be a good method for investigating the combined effect of PAH and HM.

Interaction of polycyclic aromatic hydrocarbons and heavy metals on soil enzyme.

Actions and interactions of heavy metals (cadmium, zinc and plumbum) and polycyclic aromatic hydrocarbons (PAHs) [phenanthrene, fluoranthene, benzo(a)pyrene] on the soil urease and dehydrogenase activity were studied after 49 days exposure. The experimental approach was based on the uniform design which can cut the experiment time and improve the efficiency of experiments. Data treatment was essentially based on the multiple regression technique. The results showed that the action and interaction between heavy metals and PAHs were strongly dependent on the time of pollution. The dehydrogenase exhibits more sensitive to the combined pollution than urease. The negative interaction between Zn and Cd to hydrogenase activity and the combined stimulatory activity of Phenanthrene and Benzo(a)pyrene (or fluoranthene) to soil enzyme were observed. The interactions between Zn (Cd) and phenanthrene towards urease (dehydrogenase) were positive, and the interaction between Zn and benzo(a)pyrene to urease activity was negative. This study corresponds to exploratory phase in order to reveal interaction effects of heavy metals and PAHs on the soil enzyme and then to set up more in-depth analysis to increase progressively the understanding of the ecotoxicological mechanisms involved.

Influence of phenanthrene on cadmium toxicity to soil enzymes and microbial growth.

BACKGROUND: Many contaminated sites contain a variety of toxicants. Risk assessment and the development of soil quality criteria therefore require information on the interaction among toxicants. Interactions between heavy metals are relatively well studied, but little is known about those between heavy metals and polycyclic aromatic hydrocarbons (PAHs). METHODS: 0.1 mg/kg dry soil phenanthrene alone or phenanthrene plus 10 mg/kg cadmium (Cd) were added to soil to determine the influence of phenanthrene on Cd toxicity to soil enzymes (invertase, urease, dehydrogenase and phosphatase) and microorganisms (fungi, bacteria and actinomycete) in paddy soil. RESULTS AND DISCUSSION: 0.1 mg/kg phenanthrene did not reduce the number of microorganisms. However, the addition of phenanthrene to soil with Cd enhanced the Cd toxicity to soil enzymes and microorganisms. This deleterious effect was seen to mainly affect the growth of fungi and the activity of invertase, urease and dehydrogenase. The order of combined inhibition of Cd and phenanthrene was fungi>bacteria>actinomycete. CONCLUSION: The presence of phenanthrene might enhance the toxicity of Cd to soil microorganisms. Phenanthrene can easily be used by the soil actinomycetes as a source of carbon and energy and the finding may be supportive to the development of bioremediation techniques.

Development of an ELISA for the detection of bromoxynil in water.

For development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the nitrile herbicide bromoxynil, the polyclonal antibodies raised against 2,6-dibromo-4-cyano-phenoxyacetic acid (hapten) conjugated to bovine serum albumin (BSA) by the N-hydroxysuccinimide-activated ester method. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 77 days after the primary immunization. Antiserum A2 was applied to the residual analysis of some water samples, under optimized ELISA condition, the quantitative working range was from 10 to 500 ppb with a limit of detection of 5 ppb. Cross-reactivity to structurally similar agrochemicals and related chemicals was determined. The antiserum showed little cross-reactivity with 2,6-dibromophenol and bromoxynil octanoate ester which have a dibromophenol group as common structure, but showed no cross-reactivity with other herbicides. Each water sample (river water, tap water, purified water, and bottled water) had a matrix effect and was investigated by adding Tween20 in the assay buffer. These four kinds of water samples were fortified with bromoxynil at several concentration levels and were directly analyzed with only dilution with an equal volume of antiserum solution, the mean recovery was 102.3%, and the mean coefficient of variation was 5.96%. The proposed ELISA turned out to be a powerful tool for monitoring of residual bromoxynil in water samples at trace level.

Nanocolloidal gold-based immunoassay for the detection of the N-methylcarbamate pesticide carbofuran.

Nanocolloidal gold particles were prepared and labeled to an anti-carbofuran monoclonal antibody (Mab). This conjugate was dispensed on the conjugated pad of a porous glass fiber. Ovalbumin (OVA)-carbofuran and goat anti-mouse IgG were dispensed on the nitrocellulose (NC) membrane and served as the test line and control line, respectively. The carbofuran-containing sample migrated to the NC membrane and reacted with the anti-carbofuran Mab labeled with the colloidal gold. The mixture diffused along the membrane and passed through the OVA-carbofuran in the test line via capillary action. The more analyte present in the sample, the more effectively it will compete with the carbofuran immobilized on the test line for binding to the limited amount of antibody labeled with colloidal gold. An adequate amount of carbofuran could prevent attachment of the colored conjugate to the test line. The presence or absence of a colored band on the test line could indicate a negative or positive result, respectively. When measured to the water sample spiked with carbofuran, this was obtained at or above 0.25 mg/L of carbofuran. The major advantages of the one-step strip test are that the detection time needed was <10 min and all of the reagents are included in the test device.