RESUMO
Bacteria can synthesize a broad spectrum of multifunctional polysaccharides including extracellular polysaccharides (EPS). Bacterial EPS can be utilized in the food, pharmaceutical, and biomedical areas owing to their physical and rheological properties in addition to generally presenting low toxicity. From an ecological viewpoint, EPS are biodegradable and environment compatible, offering several advantages over synthetic compounds. This study investigated the EPS produced by Klebsiella oxytoca (KO-EPS) by chemically characterizing and evaluating its properties. The monosaccharide components of the KO-EPS were determined by HPLC coupled with a refractive index detector and GC-MS. The KO-EPS was then analyzed by methylation analysis, FT-IR and NMR spectroscopy to give a potential primary structure. KO-EPS demonstrated the ability to stabilize hydrophilic emulsions with various hydrophobic compounds, including hydrocarbons and vegetable and mineral oils. In terms of iron chelation capacity, the KO-EPS could sequester 41.9 % and 34.1 % of the most common iron states, Fe2+ and Fe3+, respectively. Moreover, KO-EPS exhibited an improvement in the viscosity of aqueous dispersion, being proportional to the increase in its concentration and presenting a non-Newtonian pseudoplastic flow behavior. KO-EPS also did not present a cytotoxic effect indicating that the KO-EPS could have potential applications as a natural thickener, bioemulsifier, and bioremediation agent.
Assuntos
Biodegradação Ambiental , Emulsões , Klebsiella oxytoca , Polissacarídeos Bacterianos , Reologia , Klebsiella oxytoca/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/biossíntese , Emulsificantes/química , Emulsificantes/metabolismo , Biotecnologia/métodos , Viscosidade , Interações Hidrofóbicas e HidrofílicasRESUMO
Agrobacterium's journey has been a roller coaster, from being a pathogen to becoming a powerful biotechnological tool. While A. tumefaciens has provided the scientific community with a versatile tool for plant transformation, Agrobacterium rhizogenes has given researchers a Swiss army knife for developing many applications. These applications range from a methodology to regenerate plants, often recalcitrant, to establish bioremediation protocols to a valuable system to produce secondary metabolites. This chapter reviews its discovery, biology, controversies over its nomenclature, and some of the multiple applications developed using A. rhizogenes as a platform.
Assuntos
Agrobacterium , Biotecnologia , Agrobacterium/genética , Biotecnologia/métodos , Transformação Genética , História do Século XX , História do Século XXI , Plantas Geneticamente Modificadas/genética , Plantas/microbiologia , Plantas/genéticaRESUMO
Quartzite caves located on table-top mountains (tepuis) in the Guyana Shield, are ancient, remote, and pristine subterranean environments where microbes have evolved peculiar metabolic strategies to thrive in silica-rich, slightly acidic and oligotrophic conditions. In this study, we explored the culturable fraction of the microbiota inhabiting the (ortho)quartzite cave systems in Venezuelan tepui (remote table-top mountains) and we investigated their metabolic and enzymatic activities in relation with silica solubilization and extracellular hydrolytic activities as well as the capacity to produce antimicrobial compounds. Eighty microbial strains were isolated with a range of different enzymatic capabilities. More than half of the isolated strains performed at least three enzymatic activities and four bacterial strains displayed antimicrobial activities. The antimicrobial producers Paraburkholderia bryophila CMB_CA002 and Sphingomonas sp. MEM_CA187, were further analyzed by conducting chemotaxonomy, phylogenomics, and phenomics. While the isolate MEM_CA187 represents a novel species of the genus Sphingomonas, for which the name Sphingomonas imawarii sp. nov. is proposed, P. bryophila CMB_CA002 is affiliated with a few strains of the same species that are antimicrobial producers. Chemical analyses demonstrated that CMB_CA002 produces ditropolonyl sulfide that has a broad range of activity and a possibly novel siderophore. Although the antimicrobial compounds produced by MEM_CA187 could not be identified through HPLC-MS analysis due to the absence of reference compounds, it represents the first soil-associated Sphingomonas strain with the capacity to produce antimicrobials. This work provides first insights into the metabolic potential present in quartzite cave systems pointing out that these environments are a novel and still understudied source of microbial strains with biotechnological potential.
Assuntos
Bactérias , Cavernas , Filogenia , RNA Ribossômico 16S , Cavernas/microbiologia , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/metabolismo , Bactérias/isolamento & purificação , Bactérias/genética , Dióxido de Silício/química , Microbiota , Venezuela , Sphingomonas/metabolismo , Sphingomonas/isolamento & purificação , Sphingomonas/classificação , Sphingomonas/genética , Biotecnologia/métodos , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Microbiologia do Solo , DNA Bacteriano/genéticaRESUMO
Follicle-stimulating hormone (FSH) is an important protein used for bovine ovarian hyperstimulation in multiple ovulation and embryo transfer technology (MOET). Several attempts to produce bovine FSH (bFSH) in recombinant systems have been reported, nonetheless, up to date, the most commonly used products are partially purified preparations derived from porcine or ovine (pFSH or oFSH) pituitaries. Here we describe the development of a biotechnology process to produce a novel, hyperglycosylated, long-acting recombinant bFSH (LA-rbFSH) by fusing copies of a highly O-glycosylated peptide. LA-rbFSH and a nonmodified version (rbFSH) were produced in suspension CHO cell cultures and purified by IMAC with high purity levels (>99%). LA-rbFSH presented a higher glycosylation degree and sialic acid content than rbFSH. It also demonstrated a notable improvement in pharmacokinetic properties after administration to rats, including a higher concentration in plasma and a significant (seven-fold) reduction in apparent clearance (CLapp). In addition, the in vivo specific bioactivity of LA-rbFSH in rats was 2.4-fold higher compared to rbFSH. These results postulate this new molecule as an attractive substitute for commercially available porcine pituitary-derived products.
Assuntos
Cricetulus , Hormônio Foliculoestimulante , Proteínas Recombinantes , Animais , Hormônio Foliculoestimulante/metabolismo , Células CHO , Glicosilação , Bovinos , Ratos , Feminino , Biotecnologia/métodosRESUMO
Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.
Assuntos
Biofilmes , Dextranase , Flavobacterium , Glicosídeo Hidrolases , Streptococcus mutans , Biofilmes/crescimento & desenvolvimento , Dextranase/metabolismo , Dextranase/genética , Flavobacterium/enzimologia , Flavobacterium/genética , Streptococcus mutans/enzimologia , Streptococcus mutans/genética , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Hidrólise , Biotecnologia/métodosRESUMO
Textile and medical effluents causing bioaccumulation and biomagnification have been successfully biodegraded by fungal laccases. Here, a decision-making tool was developed and applied to evaluate 45 different laccase production strategies which determined the best potential source from a techno-economical perspective. Laccase production cost was calculated with a fixed output of 109 enzymatic units per batch (USD$per109U) and a sensitivity analysis was performed. Results indicate that optimization of enzymatic kinetics for each organism is essential to avoid exceeding the fermentation time point at which production titer reaches its peak and, therefore, higher production costs. Overall, the most cost-effective laccase-producing strategy was obtained when using Pseudolagarobasidium acaciicola with base production cost of USD $42.46 per 109 U. This works serves as platform for decision-making to find the optimal laccase production strategy based on techno-economic parameters.
Assuntos
Lacase , Lacase/metabolismo , Técnicas de Apoio para a Decisão , Biotecnologia/métodos , Biotecnologia/economia , Fungos/enzimologia , Cinética , FermentaçãoRESUMO
Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: ⢠Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments ⢠Genome-based taxonomic affiliation revealed seven potentially novel species ⢠Genome mining showed metabolic potential for novel natural products.
Assuntos
Sedimentos Geológicos , Família Multigênica , Filogenia , Microbiologia do Solo , Regiões Antárticas , Sedimentos Geológicos/microbiologia , Metabolismo Secundário/genética , Actinobacteria/genética , Actinobacteria/metabolismo , Actinobacteria/classificação , Genoma Bacteriano , Biotecnologia/métodos , Vias Biossintéticas/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismoRESUMO
In this article we can observe the scanning by the literature for the pretreatment of steam explosion applied to lignocellulose biomass. A comparison of the chemical and physical characterization of potato peel as a lignocellulose biomass. Besides, the innovative design of a continuous reactor for the potato peel steam explosion process is shown, with specific temperature and pressure conditions on a pilot scale, detailing its parts. Finally, a finite element analysis was performed where stress results were obtained from the reactor material, severity factor, structural analysis and thermal analysis, providing a panorama of the reactor's behavior with the conditions specific.
Assuntos
Solanum tuberosum , Vapor , Solanum tuberosum/química , Lignina/química , Desenho de Equipamento , Temperatura , Biomassa , Análise de Elementos Finitos , Pressão , Biotecnologia/métodos , Reatores BiológicosRESUMO
The occlusion bodies of Autographa californica multiple nucleopolyhedrovirus are proteinaceous formations with significant biotechnological potential owing to their capacity to integrate foreign proteins through fusion with polyhedrin, their primary component. However, the strategy for successful heterologous protein inclusion still requires further refinement. In this study, we conducted a comparative assessment of various conditions to achieve the embedding of recombinant proteins within polyhedra. Two baculoviruses were constructed: AcPHGFP (polh+), with GFP as a fusion to wild type (wt) polyhedrin and AcΔPHGFP (polh+), with GFP fused to a fragment corresponding to amino acids 19 to 110 of polyhedrin. These baculoviruses were evaluated by infecting Sf9 cells and stably transformed Sf9, Sf9POLH, and Sf9POLHE44G cells. The stably transformed cells contributed another copy of wt or a mutant polyhedrin, respectively. Polyhedra of each type were isolated and characterized by classical methods. The fusion PHGFP showed more-efficient incorporation into polyhedra than ΔPHGFP in the three cell lines assayed. However, ΔPHGFP polyhedron yields were higher than those of PHGFP in Sf9 and Sf9POLH cells. Based on an integral analysis of the studied parameters, it can be concluded that, except for the AcΔPHGFP/Sf9POLHE44G combination, deficiencies in one factor can be offset by improved performance by another. The combinations AcPHGFP/Sf9POLHE44G and AcΔPHGFP/Sf9POLH stand out due to their high level of incorporation and the large number of recombinant polyhedra produced, respectively. Consequently, the choice between these approaches becomes dependent on the intended application.
Assuntos
Biotecnologia , Nucleopoliedrovírus , Spodoptera , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/metabolismo , Animais , Células Sf9 , Biotecnologia/métodos , Spodoptera/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Matriz de Corpos de Inclusão , Corpos de Oclusão Virais/metabolismo , Corpos de Oclusão Virais/genética , Linhagem Celular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Phage display is an important technology to study protein-protein interaction and protein evolution, with applications in basic science and applied biotechnology, such as drug discovery and the development of targeted therapies. However, in order to be successful during a phage display screening, it is paramount to have good phage libraries. Here, we described detailed procedures to generate peptide phage display libraries with high diversity and billions of transformants.