Caracterización fenotípica y genotípica de cultivares de cacao (Theobroma cacao L.) de Dibulla, La Guajira, Colombia

In Sierra Nevada de Santa Marta, cacao plantations are comprised of commercial hybrid cultivars, and although native cacaos are found, they are not widely cultivated. Given the need to verify if these cacao varieties found in the Sierra region belong to the Criollo type genetic group, a phenotypic and genotypic characterization of cacao from the municipality Dibulla, La Guajira was carried out. For this, 11 cultivars were sampled in Mingueo. Phenotypic traits were evaluated using UPOV descriptors for cacao. The qualitative and quantitative parameters were compared through cluster and principal component analyses (PCA), and the quantitative variables through the non-parametric Mann-Whitney test. Molecular biology protocols were standardized, and the ITS region was sequenced to assess genetic relationships. From the sequences, groupings were carried out utilizing distance and phylogenetic methods. Finally, significant differences were found among the seeds (p = 0.01), and the white coloration of the cotyledon of the criollos or native stands out in contrast to the dark purple coloration of the hybrids. The cluster analysis, PCA, and sequence analysis groupings, showed differences between the group of native cacaos and commercial hybrids cultivated; in addition, native cacaos are related to the Criollo type group.En la Sierra Nevada de Santa Marta los cultivos de cacao están conformados mayoritariamente por cultivares híbridos comerciales y, aunque se encuentran cacaos nativos, estos son poco cultivados. Dada la necesidad de verificar si estos cultivares de cacao encontrados en la Sierra pertenecen al grupo genético tipo Criollo, se realizó una caracterización fenotípica y genotípica de cacaos del municipio Dibulla, La Guajira. Para esto, se muestrearon 11 cultivares en Mingueo. Los rasgos fenotípicos se evaluaron empleando descriptores UPOV para cacao. Los parámetros cualitativos y cuantitativos se cotejaron por análisis de conglomerado y análisis de componentes principales (ACP), y las variables cuantitativas se compararon a través de la prueba no paramétrica test de Mann-Whitney. Para evaluar las relaciones genéticas, se estandarizaron protocolos de biología molecular y se secuenció la región ITS. A partir de las secuencias, se realizaron agrupamientos por métodos de distancia y filogenéticos. Finalmente, se encontraron diferencias significativas entre las semillas (p = 0,01), y resalta la coloración blanca del cotiledón de los criollos en contraste con la coloración púrpura oscura de los híbridos. Asimismo, los análisis de conglomerados, ACP y los análisis de secuencias demostraron diferencias entre el grupo de los cacaos nativos y los híbridos comerciales cultivados; además, los cacaos nativos se emparentan con el grupo de cacao tipo Criollo

From Data Mining of Chitinophaga sp. Genome to Enzyme Discovery of a Hyperthermophilic Metallocarboxypeptidase.

For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications

Docking and Molecular Dynamic of Microalgae Compounds as Potential Inhibitors of Beta-Lactamase

Bacterial resistance is responsible for a wide variety of health problems, both in children and adults. The persistence of symptoms and infections are mainly treated with beta-lactam antibiotics. The increasing resistance to those antibiotics by bacterial pathogens generated the emergence of extended-spectrum beta-lactamases (ESBLs), an actual public health problem. This is due to rapid mutations of bacteria when exposed to antibiotics. In this case, beta-lactamases are enzymes used by bacteria to hydrolyze the beta-lactam rings present in the antibiotics. Therefore, it was necessary to explore novel molecules as potential beta-lactamases inhibitors to find antibacterial compounds against infection caused by ESBLs. A computational methodology based on molecular docking and molecular dynamic simulations was used to find new microalgae metabolites inhibitors of beta-lactamase. Six 3D beta-lactamase proteins were selected, and the molecular docking revealed that the metabolites belonging to the same structural families, such as phenylacridine (4-Ph), quercetin (Qn), and cryptophycin (Cryp), exhibit a better binding score and binding energy than commercial clinical medicine beta-lactamase inhibitors, such as clavulanic acid, sulbactam, and tazobactam. These results indicate that 4-Ph, Qn, and Cryp molecules, homologous from microalgae metabolites, could be used, likely as novel beta-lactamase inhibitors or as structural templates for new in-silico pharmaceutical designs, with the possibility of combatting beta-lactam resistanc

Aminopeptidase de Mesorhizobium sp. descoberta por mineração de dados genômicos com aplicações biotecnológicas e seu impacto na fisiologia bacteriana

A análise de mineração de dados genômicos de Mesorhizobium sp. revelou a presença de uma ORF de 1257pb contendo o gene mesoamp, que codifica uma proteína de 418 aminoácidos. A sequência de aminoácidos deduzida apresenta 50% de identidade com uma amino-peptidase termoestável de Thermus thermophilus, membro da família de peptidase M29, e oito assinaturas caraterísticas das metalo-protease termofílicas foram encontrados. O gene mesoamp foi clonado e expresso em Escherichia coli. A massa molecular da proteína foi avaliada por SDS-PAGE e filtração em gel, o que indicou que a proteína apresenta 45,72kDa e 88,05kDa, respectivamente, sugerindo uma estrutura dimérica da enzima recombinante. A enzima foi nomeada como MesoAmp. Em seguida realizou-se a modelagem 3D estrutural, que mostrou uma região de dimerização e uma região de ligação ao substrato altamente conservada contendo os resíduos de ligação a metais e o resíduo catalítico. A enzima apresentou atividade ótima em pH 8,5 e temperatura de 45°C, foi fortemente ativada por Co2+ e Mn2+, nestas mesmas condições de reação, a enzima apresentou Km e Kcat de 0,2364±0,018mM e 712,1±88,12seg-1, respectivamente. Além disso, foi verificado uma notável estabilidade da enzima em solventes orgânicos e elevadas concentrações de NaCl, além de um alto grau de reutilização sem perda apreciável de atividade o que torna MesoAmp única, este trabalho estabelece as bases para potenciais aplicações biotecnológicas e/ou o desenvolvimento de tecnologias sustentáveis, além de descrever uma das primeiras aminopeptidases solvente e halo-tolerantes identificados para o gênero Mesorhizobium sp.The genomic data mining analysis of a Mesorhizobium sp. revealed the presence of a 1257-bp open reading frame containing the Mesoamp gene, which encodes a protein of 418 amino acids. The deduced amino acid sequence was 50% identical to the thermostable amino-peptidase from Thermus thermophilus, a member of peptidase family M29, and eight fingerprints signatures for a thermophilic metalloprotease were found. The mesoamp gene was cloned and overexpressed in Escherichia coli. The molecular mass of the protein was assessed by SDS-PAGE and gel filtration, which indicated the protein weighs 45.72kDa and 88.05kDa, respectively, suggesting a dimeric structure of the recombinant enzyme. The enzyme was designated as MesoAmp. The 3D structural modeling showed a dimerization region with highly conserved catalytic and binding metal residues. The enzyme exhibited optimum activity at pH 8.5 and 45°C and was strongly activated by Co2+ and Mn2+ . Under these reaction conditions, the enzyme displayed Km and Kcat values of 0.2364±0.018mM and 712.1±88.12seg-1 , respectively. Additionally, the remarkable stability of the enzyme in organic solvents, its activity at high concentrations of NaCl and the high degree of reuse without appreciable loss of activity makes MesoAmp unique. In summary, this work lays the foundation for potential biotechnological applications and/or the development of environmentally friendly technologies and describes the first solvent and halo-tolerant amino-peptidases identified for the Mesorhizobium sp genus.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

From Data Mining of <i>Chitinophaga</i> sp. Genome to Enzyme Discovery of a Hyperthermophilic Metallocarboxypeptidase

For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications

Halotolerant aminopeptidase M29 from Mesorhizobium SEMIA 3007 with biotechnological potential and its impact on biofilm synthesis

The aminopeptidase gene from Mesorhizobium SEMIA3007 was cloned and overexpressed in Escherichia coli. The enzyme called MesoAmp exhibited optimum activity at pH 8.5 and 45 °C and was strongly activated by Co2+ and Mn2+. Under these reaction conditions, the enzyme displayed Km and kcat values of 0.2364 ± 0.018 mM and 712.1 ± 88.12 s−1, respectively. Additionally, the enzyme showed remarkable stability in organic solvents and was active at high concentrations of NaCl, suggesting that the enzyme might be suitable for use in biotechnology. MesoAmp is responsible for 40% of the organism’s aminopeptidase activity. However, the enzyme’s absence does not affect bacterial growth in synthetic broth, although it interfered with biofilm synthesis and osmoregulation. To the best of our knowledge, this report describes the first detailed characterization of aminopeptidase from Mesorhizobium and suggests its importance in biofilm formation and osmotic stress tolerance. In summary, this work lays the foundation for potential biotechnological applications and/or the development of environmentally friendly technologies and describes the first solvent- and halo-tolerant aminopeptidases identified from the Mesorhizobium genus and its importance in bacterial metabolism

Bg10: A Novel Metagenomics Alcohol-Tolerant and Glucose-Stimulated GH1 ß-Glucosidase Suitable for Lactose-Free Milk Preparation

<div><p>New ß-glucosidases with product (glucose) or ethanol tolerances are greatly desired to make industrial processes more marketable and efficient. Therefore, this report describes the <i>in silico</i>/<i>vitro</i> characterization of Bg10, a metagenomically derived homodimeric ß-glucosidase that exhibited a V_max of 10.81 ± 0.43 μM min^-1, K_cat of 175.1± 6.91 min^-1, and K_<i>m</i> of 0.49 ± 0.12 mM at a neutral pH and 37°C when <i>p</i>NP-ß-D-glucopyranoside was used as the substrate, and the enzyme retained greater than 80% activity within the respective pH and temperature ranges of 6.5 to 8.0 and 35 to 40°C. The enzyme was stimulated by its product, glucose; consequently, the Bg10 activity against 50 and 100 mM of glucose were increased by 36.8% and 22%, respectively, while half of the activity was retained at 350 mM. Moreover, the Bg10 was able to hydrolyse 55% (milk sample) and 100% (purified sugar) of the lactose at low (6°C) and optimum (37°C) temperatures, respectively, suggesting the possibility of further optimization of the reaction for lactose-free dairy production. In addition, the enzyme was able to fully hydrolyse 40 mM of cellobiose at one hour and was tolerant to ethanol up to concentrations of 500 mM (86% of activity), while a 1 M concentration still resulted in 41% residual activity, which could be interesting for biofuel production.</p></div

Aquifers and Groundwater: Challenges and Opportunities in Water Resource Management in Colombia

Water is essential for life on Earth, playing fundamental roles in climate regulation, ecosystem maintenance, and domestic, agricultural, and industrial processes. A total of 70% of the planet is covered by water. However, only 2.5% is fresh water, and much of it is inaccessible. Groundwater is the main source of the planet’s available water resources. For that reason, groundwater is a critically important resource, and is increasingly vulnerable due to the climate crisis and contamination. These challenges threaten the availability of clean and safe water, necessitating an understanding of effective and sustainable management. This review presents an overview of the concepts of aquifers and groundwater. Also, it reflects on the importance of these resources in developing countries such as Colombia (South America). In addition, it considers the characteristics of mineral waters, their uses, and associated risks, as well as their exploration and control policies. Colombia is a country with immense water and biological wealth and is crucial to maintaining the climate and availability of global water resources. Nevertheless, managing Colombia’s aquifers is a challenge, as many have not yet been fully explored. In order to achieve this, it is necessary to study hydrogeochemistry through the application of advanced technologies to analyze the dynamics, distribution, and quality of groundwater, as well as its vulnerability to pollution and climate change. On the other hand, the consumption of mineral groundwater can have health benefits, such as positive cardiovascular and gastrointestinal effects. But geogenic, biogenic, or anthropogenic elements such as heavy metals and microplastics can pose a risk to human health. The need for proper management of water resources to prevent risks to human health and the environment is emphasized. Therefore, an integrated approach to water resource management will ensure conservation and sustainable use, secure a continuous supply of freshwater, and facilitate adaptation to climate change

“Group I view”: Phylogenetic relationship between Bg10 and other previously characterized GH1 β-glucosidases.

<p>The tree was constructed using the Bayesian model with algorithmic tests to determine the better amino acid substitution matrix (Phangorn at “R” software) and phylogenetic model (Mr Bayes software) using two billion generations (Ngen) to find the better method. The scale bar indicates the number of amino acid substitutions per site. In this view, the focus is on the related group I. The sequence for Bg10 fell in group II. Observation: for a complete tree view, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.s001" target="_blank">S1 Fig</a>; for visualization of other groups, see also Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.g002" target="_blank">2</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167932#pone.0167932.g004" target="_blank">4</a>. Colour code: Firmicutes, dark green; Proteobacteria, pink; Thermotogales, orange; Dictyglomales, light blue; Petrotogales, brow; unculturable, purple. Numbers in white specify the amount of sequences in each collapsed branch.</p