Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of similar to 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence. (C) 2008 Elsevier Inc. All rights reserved.

Room Temperature Ionic Liquid-Lithium Salt Mixtures: Optical Kerr Effect Dynamical Measurements

The addition of lithium salts to ionic liquids causes an increase in viscosity and a decrease in ionic mobility that hinders their possible application as an alternative solvent in lithium ion batteries. Optically heterodyne-detected optical Kerr effect spectroscopy was used to study the change in dynamics, principally orientational relaxation, caused by the addition of lithium bis(trifluoromethylsulfonyl)imide to the ionic liquid 1-buty1-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. Over the time scales studied (1 ps-16 ns) for the pure ionic liquid, two temperature-independent power laws were observed: the intermediate power law (1 ps to similar to 1 ns), followed by the von Schweidler power law. The von Schweidler power law is followed by the final complete exponential relaxation, which is highly sensitive to temperature. The lithium salt concentration, however, was found to affect both power laws, and a discontinuity could be found in the trend observed for the intermediate power law when the concentration (mole fraction) of lithium salt is close to chi(LiTf(2)N) = 0.2. A mode coupling theory (MCT) schematic model was also used to fit the data for both the pure ionic liquid and the different salt concentration mixtures. It was found that dynamics in both types of liquids are described very well by MCT.

Photophysics and photostability of adenine in aqueous solution: A theoretical study

The sequential Monte Carlo/CASPT2 approach was employed to investigate deactivation and emission processes from the lowest-lying pi pi * and n pi * excited states of 9H-adenine in aqueous solution. It is found that conical intersections connecting the pi pi* and n pi* states with the ground state are also present in solution, whereas the barriers for the deactivation paths are significantly smaller on solvated conditions. The large destabilization of the n pi* state found in solution possibly prevents its involvement in the deactivation photophysics and explains the change from a bi- to a mono-exponential decay for the molecule in the gas phase and solution, respectively. (C) 2010 Elsevier B.V. All rights reserved.

Picosecond Dynamics of Proton Transfer of a 7-Hydroxyflavylium Salt in Aqueous-Organic Solvent Mixtures

The intermediacy of the geminate base proton pair (A*center dot center dot center dot H(+)) in excited-state proton-transfer (ESPT) reactions (two-step mechanism) has been investigated employing the synthetic flavylium salt 7-hydroxy-4-methyl-flavylium chloride (HMF). In aqueous solution, the ESPT mechanism involves solely the excited acid AH* and base A* forms of HMF as indicated by the fluorescence spectra and double-exponential fluorescence decays (two species, two decay times). However, upon addition of either 1,4-dioxane or 1,2-propylene glycol, the decays become triple-exponential with a term consistent with the presence of the geminate base proton pair A*center dot center dot center dot H(+). The geminate pair becomes detectable because of the increase in the recombination rate constant, k(rec), of (A*center dot center dot center dot H(+)) with increasing the mole fraction of added organic cosolvent. Because the two-step ESPT mechanism splits the intrinsic prototropic reaction rates (deprotonation of AH(+)*, k(d), and recombination, k(rec) of A*center dot center dot center dot H(+)) from the diffusion controlled rates (dissociation, k(diss) and formation, k(diff)[H(+)], of A*center dot center dot center dot H+), the experimental detection of the geminate pair provides a wealth of information on the proton-transfer reaction (k(d) and k(rec)) as well as on proton diffusion/migration (k(diss) and k(diff)).

Cellulose swelling by protic solvents: which properties of the biopolymer and the solvent matter?

The question posed in the title has been addressed by studying the swelling of celluloses at 20 C by twenty protic solvents, including water; linear- and branched-chain aliphatic alcohols; unsaturated aliphatic alcohols, and alkoxyalcohols. The biopolymers investigated included microcrystalline cellulose, MC, native and never-dried mercerized cotton cellulose, cotton and M-cotton, and native and never-dried mercerized eucalyptus cellulose, eucalyptus and M-eucalyptus, respectively. In most cases, better correlations with the physico-chemical properties of the solvents were obtained when the swelling was expressed as number of moles of solvent/anhydroglucose unit, nSw, rather than as % increase in sample weight. The descriptors employed in these correlations included, where available, Hildebrand`s solubility parameters, Gutmann`s acceptor and donor numbers, solvent molar volume, V(S), as well as solvatochromic parameters. The latter, employed for the first time for correlating the swelling of biopolymers, included empirical solvent polarity, E(T)(30), solvent ""acidity"", alpha(S), ""basicity"", beta(S), and dipolarity/polarizability, pi(S)*, respectively. Small regression coefficients and large sums of the squares of the residues were obtained when values of nSw were correlated with two solvent parameters. Much better correlations were obtained with three solvent parameters. The most statistically significant descriptor in the correlation equation depends on the cellulose, being pi(S)* for MC, cotton, and eucalyptus, and V(S) for M-cotton and M-eucalyptus. The best correlations were obtained with the same set of four parameters for all celluloses, namely, solvent pKa (or alpha(S)) beta(S), pi(S)*, and V(S), respectively. These results indicate that the supra-molecular structure of the biopolymer, in particular the average sizes of crystallites and micro-pores, and the presence of its chains in parallel (cellulose I) or anti-parallel (cellulose II) arrangements control its swelling. At least for the present biopolymer/solvent systems, use of solvatochromic parameters is a superior alternative to Hildebrand`s solubility parameters and/or Gutmann`s acceptor and donor numbers. The relevance of these results to the accessibility of the hydroxyl groups of cellulose, hence to its reactivity, is briefly discussed.

Acetylation of cellulose in LiCl-N,N-dimethylacetamide: first report on the correlation between the reaction efficiency and the aggregation number of dissolved cellulose

The acylation of three cellulose samples by acetic anhydride, Ac(2)O, in the solvent system LiCl/N,N-dimethylacetamide, DMAc (4 h, 110 A degrees C), has been revisited in order to investigate the dependence of the reaction efficiency on the structural characteristics of cellulose, and its aggregation in solution. The cellulose samples employed included microcrystalline, MCC; mercerized cotton linters, M-cotton, and mercerized sisal, M-sisal. The reaction efficiency expresses the relationship between the degree of substitution, DS, of the ester obtained, and the molar ratio Ac(2)O/AGU (anhydroglucose unit of the biopolymer); 100% efficiency means obtaining DS = 3 at Ac(2)O/AGU = 3. For all celluloses, the dependence of DS on Ac(2)O/AGU is described by an exponential decay equation: DS = DS(o) - Ae(-[(Ac2O/AGU)/B]); (A) and (B) are regression coefficients, and DS(o) is the calculated maximum degree of substitution, achieved under the conditions of each experiment. Values of (B) are clearly dependent on the cellulose employed: B((M-cotton)) > B((M-sisal)) > B((MCC)); they correlate qualitatively with the degree of polymerization of cellulose, and linearly with the aggregation number, N(agg), of the dissolved biopolymer, as calculated from static light scattering measurements: (B) = 1.709 + 0.034 N(agg). To our knowledge, this is the first report on the latter correlation; it shows the importance of the physical state of dissolved cellulose, and serves to explain, in part, the need to use distinct reaction conditions for MCC and fibrous celluloses, in particular Ac(2)O/AGU, time, temperature.

Static and dynamic bimolecular fluorescence quenching of porphyrin dendrimers in solution

The fluorescence quenching kinetics of two porphyrin dendrimer series (GnTPPH(2) and GnPZn) by different type of quenchers is reported. The microenvironment surrounding the core in GnPZn was probing by core-quencher interactions using benzimidazole. The dependence of quencher binding constant (K(a) ) on generation indicates the presence of a weak interaction between branches and the core of the porphyrin dendrimer. The similar free volume in dendrimers of third and fourth generation suggests that structural collapse in high generations occurs by packing of the dendrimer peripheral layer. Dynamic fluorescence quenching of the porphyrin core by 1,3-dicyanomethylene-2-methyl-2-pentyl-indan (PDCMI) in GnTPPH(2) is a distance dependent electron transfer process with an exponential attenuation factor beta=0.33 angstrom(-1). The quenching by 1,2-dibromobenzene occurs by diffusion process of the quencher toward to the porphyrin core, and its rate constant is practically independent of dendrimer generation.

Fluorescent probes with malononitrile side group in methyl methacrylate copolymers

Fluorescent probes derivated from auramine, 1-aminopyrene, and 9-aminoacridine containing a malononitrile group are copolymerized with methyl methacrylate. These new fluorescent polymeric materials are studied in solution of different solvents by steady-state and time-resolved emission techniques. Their spectroscopic properties and excited state dynamics are driven by charge transfer from the aromatic group to the electron withdrawing CN groups, and this factor is responsible for the non-exponential emission decay behavior. (c) 2008 Elsevier B.V. All rights reserved.

Solving the Vehicle Routing Problem with Genetic ALgorithm and Simulated Annealing

This Thesis Work will concentrate on a very interesting problem, the Vehicle Routing Problem (VRP). In this problem, customers or cities have to be visited and packages have to be transported to each of them, starting from a basis point on the map. The goal is to solve the transportation problem, to be able to deliver the packages-on time for the customers,-enough package for each Customer,-using the available resources- and – of course - to be so effective as it is possible.Although this problem seems to be very easy to solve with a small number of cities or customers, it is not. In this problem the algorithm have to face with several constraints, for example opening hours, package delivery times, truck capacities, etc. This makes this problem a so called Multi Constraint Optimization Problem (MCOP). What’s more, this problem is intractable with current amount of computational power which is available for most of us. As the number of customers grow, the calculations to be done grows exponential fast, because all constraints have to be solved for each customers and it should not be forgotten that the goal is to find a solution, what is best enough, before the time for the calculation is up. This problem is introduced in the first chapter: form its basics, the Traveling Salesman Problem, using some theoretical and mathematical background it is shown, why is it so hard to optimize this problem, and although it is so hard, and there is no best algorithm known for huge number of customers, why is it a worth to deal with it. Just think about a huge transportation company with ten thousands of trucks, millions of customers: how much money could be saved if we would know the optimal path for all our packages.Although there is no best algorithm is known for this kind of optimization problems, we are trying to give an acceptable solution for it in the second and third chapter, where two algorithms are described: the Genetic Algorithm and the Simulated Annealing. Both of them are based on obtaining the processes of nature and material science. These algorithms will hardly ever be able to find the best solution for the problem, but they are able to give a very good solution in special cases within acceptable calculation time.In these chapters (2nd and 3rd) the Genetic Algorithm and Simulated Annealing is described in details, from their basis in the “real world” through their terminology and finally the basic implementation of them. The work will put a stress on the limits of these algorithms, their advantages and disadvantages, and also the comparison of them to each other.Finally, after all of these theories are shown, a simulation will be executed on an artificial environment of the VRP, with both Simulated Annealing and Genetic Algorithm. They will both solve the same problem in the same environment and are going to be compared to each other. The environment and the implementation are also described here, so as the test results obtained.Finally the possible improvements of these algorithms are discussed, and the work will try to answer the “big” question, “Which algorithm is better?”, if this question even exists.

Hand Gesture Detection & Recognition System

The project introduces an application using computer vision for Hand gesture recognition. A camera records a live video stream, from which a snapshot is taken with the help of interface. The system is trained for each type of count hand gestures (one, two, three, four, and five) at least once. After that a test gesture is given to it and the system tries to recognize it.A research was carried out on a number of algorithms that could best differentiate a hand gesture. It was found that the diagonal sum algorithm gave the highest accuracy rate. In the preprocessing phase, a self-developed algorithm removes the background of each training gesture. After that the image is converted into a binary image and the sums of all diagonal elements of the picture are taken. This sum helps us in differentiating and classifying different hand gestures.Previous systems have used data gloves or markers for input in the system. I have no such constraints for using the system. The user can give hand gestures in view of the camera naturally. A completely robust hand gesture recognition system is still under heavy research and development; the implemented system serves as an extendible foundation for future work.