Purification Of Microbial β-galactosidase From Kluyveromyces Fragilis By Bioaffinity Partitioning

This work investigated the partitioning of β-galactosidase from Kluyveromyces fragilis in aqueous two-phase systems (ATPS) by bioaffinity. PEG 4000 was chemically activated with thresyl chloride, and the biospecific ligand p-aminophenyl 1-thio-β-D-galactopyranoside (APGP) was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme β-galactosidase from Kluyveromyces fragilis was developed. In the first step, a system composed of 6% PEG 4000-APGP and 8% dextran 505 was used, where β-galactosidase was strongly partitioned to the top phase (K = 2,330). In the second step, a system formed of 13% PEG-APGP and 9% phosphate salt was used to revert the value of the partition coefficient of β-galactosidase (K = 2 x 10-5) in order to provide the purification and recovery of 39% of the enzyme in the bottom salt-rich phase.304324331Aguiñaga-Díaz, P.A., Guzmán, R.Z., Affinity partitioning of metal ions in aqueous polyethylene glycol/salt two-phase systems with PEG-modified dictators (1996) Sep. Sci. Technol., 31 (10), pp. 1483-1499Albertsson, P.Å., (1971) Partition of Cell Particles and Macromolecules. 2nd Ed., p. 323. , New York: InterscienceAlbertsson, P.Å., Tjemeld, F., Phase Diagrams (1994) Meth. Enzumol., 228, pp. 3-13Baskir, J.N., Hatton, T.A., Suter, U.W., Protein partitioning in two-phase aqueous polymer systems (1989) Biotechol. Bioeng., 34 (4), pp. 541-558Birkenmeier, G., Partitioning of blood proteins using immobilized dyes (1994) Meth. Enzymol., 228, pp. 154-167Brena, B.M., Rydén, L.G., Porath, J., Immobilization of β-galactosidase on metal-chelate-substituted gels (1994) Biotechnol. Appl. Biochem., 19 (2), pp. 217-231Chung, B.H., Bailey, D., Arnold, F.H., Metal affinity partitioning (1994) Meth. Enymol., 228, pp. 167-179Delgado, C., Patel, J.N., Francis, G.E., Fisher, D., Coupling of poly (ethyleneglycol) to albumin under very mild conditions by activation with chloride: Characterization of the conjugate by partitioning in aqueous two-phase systems (1990) Biotechnol. Appl. Biochem., 12, pp. 110-128Diamond, A.D., Hsu, J.T., Aqueous two-phase systems for biomolecule separation (1992) Adv. Biochem. Eng./Biolechnol., 47, pp. 89-135Flores, S.H., Alegre, R.M., β-galactosidase by Erwinia aroideae grown in cheese whey (1996) Arq. Biol. Tecnol., 39 (4), pp. 879-886Franco, T.T., Andrews, A.T., Asenjo, J.A., Use of chemically modified proteins to study the effect of a single protein characteristics in aqueous two-phase systems. Effect of surface hydrophobicity (1996) Biotechnol. Bioeng., 49, pp. 300-308Godfrey, T., Reichelt, J., (1983) Industrial Enzymology, pp. 514-548. , Hampshire: MacMillian Publishers LtdGuy, E.J., Bingham, E.W., Properties of β-galactosidase of Saccharomyces lactis in milk and milk products (1978) J. Dairy Sci., 61 (2), pp. 147-151Kitahata, S., Fujita, K., Hara, K., Hashimoto, H., Enzymatic synthesis of 4-O-β-galactosyl-maltopentocose by Bacillus circulans β-galactosidase (1991) Agricult. Biolog. Chem. J., 55 (9), pp. 2433-2434Kopperschaläger, G., Affinity extraction with dye ligands (1994) Meth. Enzymol., 228, pp. 121-136Kula, M.-R., Kroner, K.H., Hustedt, H., Purification of enzymes by liquid-liquid extraction (1982) Adv. Biochem. Engin., 24, pp. 73-118Kula, M.R., Trends and future of aqueous two-phase extraction (1990) Bioseparation, 1, pp. 181-189Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (1970) Nature, 227, pp. 680-685Landman, O., Neurospora lactose. II. Enzyme formation in the standard strain (1984) Arch. Biochem. Biophys., 52, pp. 93-109Mahoney, R.R., Whitaker, J.R., Purification and physicochemical properties of β-galactosidase from Kluyveromyces fragilis (1978) J. Food Sci., 43, pp. 584-591Nilsson, K., Mosbach, K., Immobilization of ligands with organic sulphonyl chlorides (1984) Meth. Enzymol., 104, pp. 56-69Pastore, G.M., Park, Y.K., Purification and characterization of β-galactosidase from Scopulariopsis sp (1980) J. Ferm. Technol., 58 (1), pp. 79-81Prenosil, J.E., Stuker, E., Bourne, J.R., Formation of oligosaccharides during enzymatic lactose hydrolysis and their importance in a whey hydrolysis process: Part II: Experimental (1987) Biotechnol. Bioeng., 30, pp. 1026-1031Price, N.C., Stevens, L., (1989) Fundamentals of Enzymology, 2nd Ed., , Oxford: Science PublicationsSedmak, J.J., Grossberg, S.E., A rapid, sensitive, and versatile assay for protein using coomassic brilliant blue G250 (1977) Anal. Biochem., 79 (1), pp. 544-552Steers Jr., E., Cuatrecasas, P., Pollard, H.B., The purification of β-galactosidase from Escherichia coli by affinity chromatography (1971) J. Biolog. Chem., 246 (1), pp. 196-200Silva, M.E., Pellogia, C., Piza, F.A.T., Franco, T.T., Purification of three different microbial β-galactosidases by partitioning in aqueous two-phase systems (1997) Cienc. Tecnol. Alim., 17 (3), pp. 219-223Veide, A., Strandberg, L., Enfors, S.O., Extraction of β-galactosidase fused protein A in ATPS (1957) Enz. Microb. Technol., 9, pp. 730-738Wallenfels, K., Lehman, J., Malhotra, O.P., Die spezifität der β-galactosidase von Escherichia coli ML309 (1960) Biochemische Zeitschrift, 33

Mechanical and microstructural properties of redispersible polymer-gypsum composites

Studies on gypsum modified by polymers have been conducted to assess the potential of improvement in the mechanical performance, water resistance and increasing the setting time, facilitating its handling. Gypsum-based compounds made with different additions of redispersible polymers were studied, such as: ethylene-vinyl acetate (EVA), vinyl acetate terpolymer, vinyl laurate and vinyl chloride (VA/VL/VC), and vinyl acetate and vinyl versatate (VA/VeoVA). The influence on setting time, microstructural formation and on the bending performance was assessed, as well as and compression of the hardened gypsum. The composites were prepared using a polymer concentration of 5% and 10%, and water/gypsum ratio of 0.6. The addition of the polymer decreased the structural robustness and change in the microstructure. We concluded that the reduction in the amount of water through additives may allow a more complete and robust training of gypsum crystals and compounds with better mechanical performance223COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESnão te

J/psi D*D* vertex from QCD sum rules

We calculated the strong form factor and coupling constant for the $J/\psi D^* D^*$ vertex in a QCD sum rule calculation. We performed a double Borel sum rule for the three point correlation function of vertex considering both $J/\psi$ and $D^*$ mesons off--shell. The form factors obtained are very different, but they give the same coupling constant.Comment: 7 pages and 4 figures, replaced version accepted for publication in Phys. Lett.

D∗DsKD^* D_s K and Ds∗DKD_s ^* D K vertices in a QCD Sum Rule approach

We calculate the strong form factors and coupling constants of $D^* D_s K$ and $D_s^* D K$ vertices using the QCD sum rules technique. In each case we have considered two different cases for the off-shell particle in the vertex: the ligthest meson and one of the heavy mesons. The method gives the same coupling constant for each vertex. When the results for different vertices are compared, they show that the SU(4) symmetry is broken by around 40%.Comment: 10 pages, 8 figures. Submitted to Phys. Lett.

Model-based estimation of transmissibility and reinfection of SARS-CoV-2 P.1 variant

Acknowledgements We are grateful for the collaborative work of the reviewers and the entire group of the Observatório COVID-19 BR. In particular, we thank Verônica Coelho for critical inputs. We also thank the research funding agencies: the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (Finance Code 001 to F.M.D.M., L.S.F. and T.P.P.), Conselho Nacional de Desenvolvimento Científico e Tecnológico—Brazil (grant number: 315854/2020-0 to M.E.B., 141698/2018-7 to R.L.P.S., 313055/2020-3 to P.I.P., 312559/2020-8 to M.A.S.M.V., 311832/2017-2 to R.A.K., 305703/2019-6 to A.A.M.S.) and Fundação de Amparo à Pesquisa do Estado de São Paulo—Brazil (grant number: 2019/26310-2 and 2017/26770-8 to C.F., 2018/26512-1 to O.C., 2018/24037-4 to S.P. and contract number: 2016/01343-7 to R.A.K.). The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers of Disease Control and Prevention.Peer reviewe

Esr Of Trapped Centers In γ Irradiated Silver Chlorates Single Crystals

The paramagnetic centers O3-, ClO2, and (ClO2-Cl)- have been identified in irradiated AgClO 3 single crystals by X and Q band ESR experiments at room temperatures and by the optical absorption bands at 480 and 360 nm, characteristic of the first two centers, respectively. The ESR spectrum and its angular dependence show that the first two centers have two magnetically inequivalent sites per unit cell. The third center was clearly observed only at certain orientations. The data for ClO2 show that the principal directions of the g and A tensors do not coincide; the two magnetically inequivalent sites have similar principal values of the g and A tensors related by a 90° rotation about the tetragonal crystal axis of symmetry, and can therefore be assumed to be chemically equivalent. It is found that the ClO 2 center is formed substitutionally in the ClO3- ion position, but that the ozonide O3- is not. The thermal annealing of the γ irradiated AgClO3 has revealed an increase in the concentration of the O3- radical that seems to be connected with the simultaneous destruction of the ClO2 center. Copyright © 1976 American Institute of Physics.6593461346

Bs∗BKB_s^* B K vertex from QCD sum rules

The form factors and the coupling constant of the $B_s^* B K$ vertex are calculated using the QCD sum rules method. Three point correlation functions are computed considering both $K$ and $B$ mesons off-shell and, after an extrapolation of the QCDSR results, we obtain the coupling constant of the vertex. We study the uncertainties in our result by calculating a third form factor obtained when the $B^*_s$ is the off-shell meson, considering other acceptable structures and computing the variations of the sum rules' parameters. The form factors obtained have different behaviors but their simultaneous extrapolations reach to the same value of the coupling constant $g_{B_s^* B K}=10.6 \pm 1.7$. We compare our result with other theoretical estimates.Comment: 11 pages, 11 figure

Activated Random Walkers: Facts, Conjectures and Challenges

We study a particle system with hopping (random walk) dynamics on the integer lattice $\mathbb Z^d$. The particles can exist in two states, active or inactive (sleeping); only the former can hop. The dynamics conserves the number of particles; there is no limit on the number of particles at a given site. Isolated active particles fall asleep at rate $\lambda > 0$, and then remain asleep until joined by another particle at the same site. The state in which all particles are inactive is absorbing. Whether activity continues at long times depends on the relation between the particle density $\zeta$ and the sleeping rate $\lambda$. We discuss the general case, and then, for the one-dimensional totally asymmetric case, study the phase transition between an active phase (for sufficiently large particle densities and/or small $\lambda$) and an absorbing one. We also present arguments regarding the asymptotic mean hopping velocity in the active phase, the rate of fixation in the absorbing phase, and survival of the infinite system at criticality. Using mean-field theory and Monte Carlo simulation, we locate the phase boundary. The phase transition appears to be continuous in both the symmetric and asymmetric versions of the process, but the critical behavior is very different. The former case is characterized by simple integer or rational values for critical exponents ($\beta = 1$, for example), and the phase diagram is in accord with the prediction of mean-field theory. We present evidence that the symmetric version belongs to the universality class of conserved stochastic sandpiles, also known as conserved directed percolation. Simulations also reveal an interesting transient phenomenon of damped oscillations in the activity density

\rho D^*D^* vertex from QCD sum rules

We calculate the form factors and the coupling constant in the $\rho D^* D^*$ vertex in the framework of QCD sum rules. We evaluate the three point correlation functions of the vertex considering both $\rho$ and $D^*$ mesons off--shell. The form factors obtained are very different but give the same coupling constant: $g_{\rho D^* D^*} = 6.6 \pm 0.31$. This number is 50% larger than what we would expect from SU(4) estimates.Comment: 9 pages, 6 eps figure

Sandpiles with height restrictions

We study stochastic sandpile models with a height restriction in one and two dimensions. A site can topple if it has a height of two, as in Manna's model, but, in contrast to previously studied sandpiles, here the height (or number of particles per site), cannot exceed two. This yields a considerable simplification over the unrestricted case, in which the number of states per site is unbounded. Two toppling rules are considered: in one, the particles are redistributed independently, while the other involves some cooperativity. We study the fixed-energy system (no input or loss of particles) using cluster approximations and extensive simulations, and find that it exhibits a continuous phase transition to an absorbing state at a critical value zeta_c of the particle density. The critical exponents agree with those of the unrestricted Manna sandpile.Comment: 10 pages, 14 figure