Mônica Rosas da Costa Iemma

Isolation of Arginase Inhibitors from the Bioactivity-Guided Fractionation of Byrsonima coccolobifolia Leaves and Stems

Byrsonima coccolobifolia leaf and stem extracts were studied in the search for possible leishmanicidal compounds using arginase (ARG) from Leishmania amazonensis as a molecular target. Flavonoids 1b, 1e-1g, 2a, 2b, and 2d-2f showed significant inhibitory activity, with IC50 values ranging from 0.9 to 4.8 μM. The kinetics of the most active compounds were determined. Flavonoids 1e, 1f, 2a, 2b, and 2e were characterized as noncompetitive inhibitors of ARG with dissociation constants (Ki) ranging from 0.24 to 3.8 μM, demonstrating strong affinity. Structure-activity relationship studies revealed some similarities in the structural features of flavonoids related to ARG activity.

Enhanced production of recombinant thermo-stable lipase in Escherichia coli at high induction temperature.

Thermostable microbial lipases are potential candidates for industrial applications such as specialty organic syntheses as well as hydrolysis of fats and oils. In this work, basic biochemical engineering tools were applied to enhance the production of BTL2 lipase cloned in Escherichia coli BL321 under control of the strong temperature-inducible λP(L) promoter. Initially, surface response analysis was used to assess the influence of growth and induction temperatures on enzyme production, in flask experiments. The results showed that temperatures of 30 and 45°C were the most suitable for growth and induction, respectively, and led to an enzyme specific activity of 706,000 U/gDCW. The most promising induction conditions previously identified were validated in fed-batch cultivation, carried out in a 2L bioreactor. Specific enzyme activity reached 770,000 U/gDCW, corresponding to 13,000 U/L of culture medium and a lipase protein concentration of 10.8 g/L. This superior performance on enzyme production was a consequence of the improved response of λP(L) promoter triggered by the high induction temperature applied (45°C). These results point out to the importance of taking into account protein structure and stability to adequately design the recombinant protein production strategy for thermally induced promoters.

Evaluating kinetic and physiological features of rCHO-K1 cells cultured on microcarriers for production of a recombinant metalloprotease/disintegrin

We present kinetic and physiological data regarding the culturing of rCHO-K1 cells on various microcarriers, to evaluate the potential of this culture strategy for mass production of these cells and expression of a recombinant disintegrin. Cultures were performed in 500 mL spinner flasks in DMEM culture medium with 10% v/v fetal calf serum, gently shaken at 37oC, pH 7.4, in a 10% v/v CO 2 atmosphere. The following values were obtained, respectively, for the adhesion time-constant Ka (h) and specific growth rate μ max (d -1 ) on each microcarrier: Cytodex 1 (0.91, 0.45), Cultispher S (0.28, 0.34), Immobasil FS (0.85, 0.52) and Pronectin F (5.12, 0.67). Metabolic characteristics showed some variation among the cultures with the four microcarriers, the most significant being the higher production of ammonia with microcarriers coated with adhesive molecules (Cultispher S and Pronectin F) relative to the uncoated carriers (Cytodex 1 and Immobasil FS). Experiments where the DMEM medium was gradually replaced by the serum-free medium (CHO-SFM-II) revealed important advantages over media containing serum, not only for assay purposes, but also for purification of the disintegrin. Altogether these results demonstrate that cultures on microcarriers, especially on Pronectin F, show good potential for larger scale cultures of rCHO-K1 cell.

Immobilization of pectinase from Leucoagaricus gongylophorus on magnetic particles.

Polygalacturonases (EC 3.2.1.15) hydrolyze the α-1,4-glycosidic linkages in polygalacturonic acid chains. The interest on specific inhibitors of pectinase and the versatility of magnetic support for enzyme immobilization endorsed the preparation of an immobilized enzyme reactor (IMER). This work presents the synthesis of CoFe(2)O(4) amino-derivatives, which was employed as the support for the immobilization of pectinases from Leucoagaricus gongylophorus. Amino-functionalized CoFe(2)O(4) was obtained from glyceryl-derivatized CoFe(2)O(4) and was characterized by infrared spectroscopy and electronic microscopy. The immobilized enzyme maintained the same thermal, chemical and kinetic behaviour of the free enzyme (T(opt) 60 °C; pH(opt) 5.0; K(app)(M) = 0.5 mg min(-1); V(app)(M) ≈ 5.0 μmol min(-1) mL(-1)). The straightforward synthesis of CoFe(2)O(4) derivatives and the efficiency of immobilization offer wide perspectives for the use of the developed new IMER.

Leishmanicidal galloylquinic acids are noncompetitive inhibitors of arginase

Leishmanicidal galloylquinic acids were isolated from the ethyl acetate extract of Byrsonima coccolobifolia. These phenols and gallic acid showed to be a new class of potent noncompetitive inhibitors of arginase ARG (Ki ranging from 0.10 to 0.68 µmol L -1 ) from Leishmania amazonensis. Quinic acid did not exhibit significant inhibition of ARG, indicating that galloyl moiety has important features that allows the enzyme-inhibitor interactions. The significant inhibitory activity of gallic acid on ARG can be a clue to understand the immune response previously observed on L. donovani, since ARG activity is associated with the decrease of the levels of NO in Leishmania infection.

Assessment of the metabolism of different strains of Bacillus megaterium

Bacillus megaterium is a promising host for expression of heterologous proteins. This paper reports the nutrient consumption patterns and production of metabolites for three different strains of B. megaterium, ATCC 14945, QMB 1551 and PV 361, which is QMB 1551 with seven constitutive plasmids deleted. 14 h cultivations in agitated flasks were run, for two different media: A (LB plus 10g/L glucose) and B (medium A, with the yeast extract replaced by tryptone). Strains PV361 and QMB 1551 showed higher maximum specific growth rates in medium B, reaching 0.42 h-1 and 0.48 h-1 respectively. The main by-products of the glucose overflow mechanism were acetate and lactate, for all three strains, which had preferential amino acids for substrate: Ala, Asp, Glu, Ser. No production of alcohols was detected.