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

Adaptabilidade, estabilidade e resistência a patógenos em genótipos de feijoeiro

Resumo:O objetivo deste trabalho foi avaliar a resistência de genótipos de feijoeiro aos principais patógenos da cultura, bem como a adaptabilidade e a estabilidade de produção de grãos desses genótipos. Avaliaram-se 26 genótipos de feijoeiro quanto à resistência a Colletotrichum lindemuthianum, Fusarium oxysporum f. sp. phaseolie Xanthomonas axonopodis pv. phaseoli, por meio de inoculação, em laboratório, e em 19 ensaios de valor de cultivo e uso (VCU), em diferentes locais do Estado de São Paulo, nas safras das "águas", "seca" e "inverno", durante os anos agrícolas 2011, 2012 e 2013. Dezoito genótipos foram considerados resistentes: sete deles a C. lindemuthianum, sete a F. oxysporumf. sp. phaseolie quatro a X. axonopodispv. phaseoli. A reação de resistência aos patógenos está associada à estabilidade dos genótipos. Por meio das análises GGE biplot, foi possível identificar genótipos com adaptabilidade e estabilidade superiores às das testemunhas, nos dois grupos de tegumento avaliados, em todas as épocas de semeadura