An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania

Influence of nicotine and diphenylamine on the carotenoid composition of Rhodotorula strains

In order to obtain commercially interesting carotenoid profiles, the effect of supplementation with diphenylamine (DPA) and nicotine in the culture media of Rhodotorula rubra and Rhodotorula glutinis was studied. Fermentation experiments were carried out in flasks. The carotenoids were extracted with ethyl acetate by physical disruption, and analyzed by high-performance liquid chromatography using a reversed-phase column. At subinhibitory levels, both the inhibitors showed no effect on yeast growth for the two strains. Only R. rubra produced more carotenoids when 5 mu mol of DPA was added to the broth, 155.8 mu g/g of dry weight. For both strains, supplementation with 5 mu mol DPA caused a significant reduction in the torularhodin/torulene ratio, from 1.5 to 0.5 for R. rubra, and from 0.8 to 0.5 for R. glutinis. With 10 mu mol of this inhibitor, an accumulation of beta-carotene occurred from 13 to 47% in R. rubra, and from 36 to 43% in R. glutinis. Supplementation with nicotine resulted in the selective biosynthesis of lycopene (up to 93.8 mu g/g) and the formation of a minor carotenoid, tentatively identified as 3,4-didehydrolycopene, not previously related to Rhodotorula spp.29663865

Discovery, structural characterization, and functional insights into a novel apiosidase from the GH140 family, isolated from a lignocellulolytic-enriched mangrove microbial community

\ua9 2024, The Author(s), under exclusive licence to Springer Nature B.V.Objectives: Apiosidases are enzymes that cleave the glycosidic bond between the monosaccharides linked to apiose, a branched chain furanose found in the cell walls of vascular plants and aquatic monocots. There is biotechnological interest in this enzyme group because apiose is the flavor-active compound of grapes, fruit juice, and wine, and the monosaccharide is found to be a plant secondary metabolite with pharmaceutical properties. However, functional and structural studies of this enzyme family are scarce. Recently, a glycoside hydrolase family member GH140 was isolated from Bacteroides thetaiotaomicron and identified as an endo-apiosidase. Results: The structural characterization and functional identification of a second GH140 family enzyme, termed MmApi, discovered through mangrove soil metagenomic approach, are described. Among the various substrates tested, MmApi exhibited activity on an apiose-containing oligosaccharide derived from the pectic polysaccharide rhamnogalacturonan-II. While the crystallographic model of MmApi was similar to the endo-apiosidase from Bacteroides thetaiotaomicron, differences in the shape of the binding sites indicated that MmApi could cleave apioses within oligosaccharides of different compositions. Conclusion: This enzyme represents a novel tool for researchers interested in studying the physiology and structure of plant cell walls and developing biocatalytic strategies for drug and flavor production

Aspergillus niger beta-Glucosidase Has a Cellulase-like Tadpole Molecular Shape INSIGHTS INTO GLYCOSIDE HYDROLASE FAMILY 3 (GH3) beta-GLUCOSIDASE STRUCTURE AND FUNCTION

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Background: -Glucosidase completes cellulose enzymatic hydrolysis by releasing glucose from cellobiose. Results: SAXS experiments revealed that Aspergillus niger -glucosidase has a cellulase-like tadpole molecular shape, uncommon to enzymes that act on the soluble substrates. Conclusion: We show that AnBgl1 N- and C-terminal domains are linked by a long extended linker. Significance: Understanding AnBgl1 architecture is useful for comprehension of the enzyme-cell wall interaction and the process of biomass saccharification. Aspergillus niger is known to secrete large amounts of -glucosidases, which have a variety of biotechnological and industrial applications. Here, we purified an A. niger -glucosidase (AnBgl1) and conducted its biochemical and biophysical analyses. Purified enzyme with an apparent molecular mass of 116 kDa forms monomers in solution as judged by native gel electrophoresis and has a pI value of 4.55, as found for most of the fungi of -glucosidases. Surprisingly, the small angle x-ray experiments reveal that AnBgl1 has a tadpole-like structure, with the N-terminal catalytic domain and C-terminal fibronectin III-like domain (FnIII) connected by the long linker peptide (approximate to 100 amino acid residues) in an extended conformation. This molecular organization resembles the one adopted by other cellulases (such as cellobiohydrolases, for example) that frequently contain a catalytic domain linked to the cellulose-binding module that mediates their binding to insoluble and polymeric cellulose. The reasons why AnBgl1, which acts on the small soluble substrates, has a tadpole molecular shape are not entirely clear. However, our enzyme pulldown assays with different polymeric substrates suggest that AnBgl1 has little or no capacity to bind to and to adsorb cellulose, xylan, and starch, but it has high affinity to lignin. Molecular dynamics simulations suggested that clusters of residues located in the C-terminal FnIII domain interact strongly with lignin fragments. The simulations showed that numerous arginine residues scattered throughout the FnIII surface play an important role in the interaction with lignin by means of cation- stacking with the lignin aromatic rings. These results indicate that the C-terminal FnIII domain could be operational for immobilization of the enzyme on the cell wall and for the prevention of unproductive binding of cellulase to the biomass lignin.288463299133005Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Instituto Nacional de Ciencia e Tecnologia do BioetanolFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [08/56255-9, 09/54035-4, 10/16542-9]CNPq [490022/2009-0

Substrate cleavage pattern, biophysical characterization and low-resolution structure of a novel hyperthermostable arabinanase from Thermotoga petrophila

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Arabinan is a plant structural polysaccharide degraded by two enzymes: alpha-L-arabinofuranosidase and endo-1,5-alpha-L-arabinanase. These enzymes are highly diversified in nature, however, little is known about their biochemical and biophysical properties. We have characterized a novel arabinanase (AbnA) isolated from Thermotoga petrophila with unique thermostable properties such as the insignificant decrease of residual activity after incubation up to 90 degrees C. We determined the AbnA mode of operation through capillary zone electrophoresis, which accumulates arabinotriose and arabinobiose as end products after hydrolysis of arabinan-containing polysaccharides. Spectroscopic analyses by Far-UV circular dichroism and intrinsic tryptophan fluorescence emission demonstrated that AbnA is folded and formed mainly by beta-sheet structural elements. In silico molecular modeling showed that the AbnA structure encompasses a five-bladed beta-propeller catalytic core juxtaposed by distorted up-and-down beta-barrel domain. The low-resolution structure determined by small angle X-ray scattering indicated that AbnA is monomeric in solution and its molecular shape is in full agreement with the model. (C) 2010 Elsevier Inc. All rights reserved.3994505511Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Department of Energy [06103-OKL, ZDJ-7-77608-01]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [08/58037-9]CNPq [478059/2009-4]Department of Energy [06103-OKL, ZDJ-7-77608-01

Characterization of a Hexameric Exo-Acting GH51 alpha-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)alpha-l-Arabinofuranosidases (alpha-l-Abfases, EC 3.2.1.55) display a broad specificity against distinct glycosyl moieties in branched hemicellulose and recent studies have demonstrated their synergistic use with cellulases and xylanases for biotechnological processes involving plant biomass degradation. In this study, we examined the structural organization of the arabinofuranosidase (GH51 family) from the mesophilic Bacillus subtilis (AbfA) and its implications on function and stability. The recombinant AbfA showed to be active over a broad temperature range with the maximum activity between 35 and 50 A degrees C, which is desirable for industrial applications. Functional studies demonstrated that AbfA preferentially cleaves debranched or linear arabinan and is an exo-acting enzyme producing arabinose from arabinoheptaose. The enzyme has a canonical circular dichroism spectrum of alpha/beta proteins and exhibits a hexameric quaternary structure in solution, as expected for GH51 members. Thermal denaturation experiments indicated a melting temperature of 53.5 A degrees C, which is in agreement with the temperature-activity curves. The mechanisms associated with the unfolding process were investigated through molecular dynamics simulations evidencing an important contribution of the quaternary arrangement in the stabilization of the beta-sandwich accessory domain and other regions involved in the formation of the catalytic interface of hexameric Abfases belonging to GH51 family.553260267Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2008/58037-9, 10/51890-8, 2011/14200-6, 2011/13242-7, 2010/11499-1]CNPq [133394/2011-5, 475022/2011-4, 310177/2011-1, 2011/17658-3, 140420/2009-6

Simultaneous production of xylooligosaccharides and antioxidant compounds from sugarcane bagasse via enzymatic hydrolysis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Advances in industrial biotechnology offer potential opportunities for economic utilization of agroindustrial residues such as sugarcane bagasse, which is the major by-product of the sugarcane industry. Due to its abundant availability and despite the complex chemical composition, it can be considered an ideal substrate for microbial processes for the production of value-added products. In the present study we evaluated the enzymatic production of xylooligosaccharides (XOS) and antioxidant compounds from sugarcane bagasse using XynZ from Clostridium thermocellum, a naturally chimeric enzyme comprising activities of xylanase and feruloyl esterase along with a carbohydrate binding module (CBM6). In order to reveal the biotechnological potential of XynZ, the XOS released after enzymatic hydrolysis using different substrates were characterized by capillary electrophoresis and quantified by high performance anion exchange chromatography. In parallel, the antioxidant capacity related to the release of phenolic compounds was also determined. The results indicated noteworthy differences regarding the amount of XOS and antioxidant phenolic compounds produced, as well as the XOS profile, functions of the pre-treatment method employed. The ability of XynZ to simultaneously produce xylooligosaccharides, natural probiotics, phenolic compounds and antioxidant molecules from natural substrates such as sugarcane bagasse demonstrated the biotechnological potential of this enzyme. Production of value-added products from agro-industrial residues is of great interest not only for advancement in the biofuel field, but also for pharmaceutical and food industries. (C) 2013 Elsevier B.V. All rights reserved.52770775Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq [474022/2011-4, 310177/2011-1, 142685/2010-0]FAPESP [2008/58037-9, 2011/14200-6, 2012/18859-5, 2013/03061-0