Assessments on the catalytic and kinetic properties of beta-glucosidase isolated from a highly efficient antagonistic fungus Trichoderma harzianum / Avaliações das propriedades catalíticas e cinéticas da beta-glucosidase isoladas de um fungo antagonísta altamente eficiente Trichoderma harzianum

Due to the toxicity and inefficiency of chemical fungicides to control infestation of Macrophomina phaseolina (Tassi) Goid which causes charcoal rot in plants, a biotechnological approach using - glucosidase (EC.3.2.1) as the alternative bioactive ingredient in fungicide is hereby, proposed. The extracellular enzyme was isolated from a highly efficient fungal antagonist, Trichoderma harzianum T12. The highly similar molecular masses obtained using SDS-PAGE (96 kDa) and MALDI-TOF mass spectrometry (98.3 kDa) affirmed that the -glucosidase was purified to homogeneity. Consequently, optimum catalytic parameters that rendered the highest enzyme activity were found to be: 45°C, pH 7, inoculum size of 10 % (w/v), supplementation with metal ions Zn2+ and Mn2+ ions, and Tween 80. Addition of wheat bran and (NH4)2SO4 as carbon and nitrogen sources also improved enzyme activity. BLASTn showed the sequence of -glucosidase T12 was highly identical to other -glucosidases viz. T. harzianum strain IOC-3844 (99%), T. gamsii and T. virens bgl1 (86 %) as well as T. reesei strain SJVTR and T. viride strain AS 3.3711 (84 %). Kinetic assessment showed that -glucosidase T12 catalyzes hydrolytic activity is characterized by a Km of 0.79 mM and Vmax of 8.45 mM min-1 mg-1 protein, with a corresponding kcat of 10.69 s-1.

Devido à toxicidade e ineficiência dos fungicidas químicos para controlar a infestação de Macrophomina phaseolina (Tassi) Goid que causa o apodrecimento das plantas, uma abordagem biotecnológica usando - glicosidase (EC.3.2.1) como o ingrediente bioativo alternativo do fungicida é por este meio, proposto. A enzima extracelular foi isolada de um antagonista fúngico altamente eficiente, o Trichoderma harzianum T12. As massas moleculares altamente similares obtidas usando SDS-PAGE (96 kDa) e espectrometria de massa MALDI-TOF (98,3 kDa) afirmaram que a -glicosidase foi purificada até a homogeneidade. Consequentemente, os parâmetros catalíticos ótimos que apresentaram a maior atividade enzimática foram: 45°C, pH 7, tamanho do inóculo de 10% (p / v), suplementação com íons de metais Zn2+ e Mn2+, e Tween 80. Adição de farelo de trigo e (NH4) 2SO4 como fontes de carbono e nitrogênio também melhoraram a atividade enzimática. O BLASTn mostrou que a sequência da -glicosidase T12 era altamente idêntica a outras -glicosidase viz. A estirpe T. harzianum IOC-3844 (99%), T. gamsii e T. virens bgl1 (86%) assim como a estirpe T. reesei SJVTR e a estirpe T. viride AS 3.3711 (84%). A avaliação cinética mostrou que -glicosidase T12 catalisa a actividade hidrolítica caracterizada por um Km de 0,79 mM e Vmax de 8,45 mM min-1 mg-1 de proteína, com um correspondente kcat de 10,69 s-1.

Glutamine treatment attenuates hyperglycemia-induced mitochondrial stress and apoptosis in umbilical vein endothelial cells

OBJECTIVE: The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis, mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical vein endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells were grown and subjected to glutamine and insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and the production of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicity assays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. RESULTS: Glutamine maintains the integrity of the mitochondria by reducing the cell permeability and cytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level was significantly (p<0.005) reduced when the cells were treated with glutamine. An apoptosis assay revealed significantly reduced apoptosis (p<0.005) in the glutamine-treated cells. Moreover, glutamine alone or in combination with insulin modulated inflammatory cytokine levels. Interleukin-10, interleukin-6, and vascular endothelial growth factor were up-regulated while tumor necrosis factor-α was down-regulated after treatment with glutamine. CONCLUSION: Glutamine, either alone or in combination with insulin, can positively modulate the mitochondrial stress and cell permeability in umbilical vein endothelial cells. Glutamine regulates the expression of inflammatory cytokines and maintains the balance of the mitochondria in a cytoprotective manner. .