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Brazilian Journal of Microbiology :... Dec 2021Yeast isolates from flowers and fruits from a Brazilian forest were studied. The yeasts were identified at species and strain level by PCR-RFLP and PCR-RAPD,...
Yeast isolates from flowers and fruits from a Brazilian forest were studied. The yeasts were identified at species and strain level by PCR-RFLP and PCR-RAPD, respectively. The 46 isolated yeasts were classified into 11 different species belonging to the genera Candida, Diutina, Hanseniaspora, Meyerozyma, Pichia, Rhodotorula, and Torulaspora. A total of 20 different strains were found. In order to ascertain the probiotic potential, the resistance to gastrointestinal conditions, autoaggregation, and hydrophobicity assays were studied, along with the capacity to form biofilm. The results indicate that, although most of the strains presented better results than Saccharomyces boulardii (the only strain recognized as a probiotic yeast), four strains were the most promising, namely, Rhodotorula mucilaginosa 32, Meyerozyma caribbica 35, and Diutina rugosa 12 and 45, according to the Duncan test. Several biotechnological properties were evaluated. D. rugosa inhibited Dekkera bruxellensis. The assimilation or fermentation of seven sugars was tested, and only five of the yeasts did not show a capacity to assimilate any of the sugars under aerobic conditions. However, all strains were able to ferment at least one of the sugars under anaerobic conditions. As far as enzyme production is concerned, positive results were only found for the enzymes' amylase, pectinase, and protease. D. rugosa 42 and Hanseniaspora opuntiae 18, followed of Pichia kluyveri 26, showed high values for the production of melatonin. In conclusion, the results of this study show that several non-Saccharomyces present probiotic characteristics, and these have good potential for industrial applications in the food or biotechnology industries.
Topics: Biotechnology; Ecosystem; Fermentation; Fruit; Probiotics; Random Amplified Polymorphic DNA Technique; Sugars; Yeasts
PubMed: 34595728
DOI: 10.1007/s42770-021-00541-z -
Biomolecules Jun 2021Water content is an important factor in lipase-catalyzed reactions in organic media but is frequently ignored in the study of lipases by molecular dynamics (MD)...
Water content is an important factor in lipase-catalyzed reactions in organic media but is frequently ignored in the study of lipases by molecular dynamics (MD) simulation. In this study, lipase B, lipase and lipase were used as research models to explore the mechanisms of lipase in micro-aqueous organic solvent (MAOS) media. MD simulations indicated that lipases in MAOS systems showed unique conformations distinguished from those seen in non-aqueous organic solvent systems. The position of water molecules aggregated on the protein surface in MAOS media is the major determinant of the unique conformations of lipases and particularly impacts the distribution of hydrophilic and hydrophobic amino acids on the lipase surface. Additionally, two maxima were observed in the water-lipase radial distribution function in MAOS systems, implying the formation of two water shells around lipase in these systems. The energy landscapes of lipases along solvent accessible areas of catalytic residues and the minimum energy path indicated the dynamic open states of lipases in MAOS systems differ from those in other solvent environments. This study confirmed the necessity of considering the influence of the microenvironment on MD simulations of lipase-catalyzed reactions in organic media.
Topics: Basidiomycota; Cellular Microenvironment; Computational Biology; Fungal Proteins; Hydrophobic and Hydrophilic Interactions; Lipase; Molecular Dynamics Simulation; Protein Structure, Secondary; Protein Structure, Tertiary; Saccharomycetales; Water
PubMed: 34200257
DOI: 10.3390/biom11060848 -
Biochimica Et Biophysica Acta.... Jul 2021Bioaugmentation is a promising method of the remediation of soils polluted by persistent organic pollutants (POP). Unfortunately, it happens frequently that the...
Bioaugmentation is a promising method of the remediation of soils polluted by persistent organic pollutants (POP). Unfortunately, it happens frequently that the microorganisms inoculated into the soil die out due to the presence of enzymes secreted by autochthonous microorganisms. Especially destructive are here phospholipases C (PLC) and lipases which destruct the microorganism's cellular membrane. The composition of bacterial membranes differs between species, so it is highly possible that depending on the membrane constitution some bacteria are more resistant to PLCs and lipases than other. To shed light on these problems we applied phospholipid Langmuir monolayers as model microbial membranes and studied their interactions with α-toxin (model bacterial PLC) and the lipase isolated from soil fungus Candida rugosa. Membrane phospholipids differing in their headgroup (phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols and cardiolipins) and in their tail structure were applied. The monolayers were characterized by the Langmuir technique, visualized by Brewster angle microscopy, and the packing mode of the phospholipid molecules was verified by the application of the diffraction of synchrotron radiation. We also studied the mutual miscibility of diacylglycerols and the native phospholipids as their interaction is crucial for the understanding of the PLC and lipase activity. It turned out that all the investigated phospholipid classes can be hydrolyzed by PLC; however, they differ profoundly in the hydrolysis degree. Depending on the effects of the initial PLC action and the mutual organization of the diacylglycerol and phospholipid molecules the lipase can ruin the model membranes or can be completely neutral to them.
Topics: Clostridium perfringens; Lipase; Models, Biological; Phosphatidylcholines; Phospholipids; Saccharomycetales; Type C Phospholipases; Unilamellar Liposomes
PubMed: 33831405
DOI: 10.1016/j.bbamem.2021.183620 -
Bioorganic Chemistry Apr 2021Candida rugosa lipase (CRL) is an enzyme commonly used in medicinal and biotechnological applications. Allosteric modulators of CRL could aid in modifying lipase-related...
Candida rugosa lipase (CRL) is an enzyme commonly used in medicinal and biotechnological applications. Allosteric modulators of CRL could aid in modifying lipase-related diseases as well as improving biotechnological processes. Thus, a combinatorial approach of computational in-silico and high-throughput in-vitro screening was used to identify allosteric modulators of CRL. The screening of natural product libraries resulted in 132 compounds of which 53 were tested in-vitro. Subsequently, four inhibitors and three enhancers were identified of which rutin and cynaroside represented the strongest inhibitors of CRL activity (IC50: 227 ± 26 µM and 446 ± 15 µM, respectively) and NP-008496 the strongest enhancer (EC50: 425 ± 18 µM). All three compounds were predicted to bind the same allosteric site suggesting a common mechanism. Therefore, the present study demonstrated a reliable work-flow, identified an allosteric site of CRL and determined inhibitors and enhancers with numerous potential medical and biotechnological applications.
Topics: Allosteric Site; Biological Products; Dose-Response Relationship, Drug; Enzyme Inhibitors; Kinetics; Lipase; Molecular Docking Simulation; Molecular Structure; Saccharomycetales; Structure-Activity Relationship
PubMed: 33639364
DOI: 10.1016/j.bioorg.2021.104732 -
Journal of Chemical Information and... Feb 2021Lipases are enzymes able to catalyze the hydrolysis or synthesis of triglycerides, depending on the reaction conditions, whereas sterol esterases show the same ability...
Lipases are enzymes able to catalyze the hydrolysis or synthesis of triglycerides, depending on the reaction conditions, whereas sterol esterases show the same ability on sterol esters. Structurally, both kinds of enzymes display an α/β-hydrolase fold, with a substrate-binding pocket formed by a hydrophobic cavity covered by a mobile lid. However, it has been reported that some lipases from the -like family display wide substrate specificity on both triglycerides and sterol esters. Among them, enzymes with different biotechnological applications, such as the lipase isoenzymes produced by and the sterol esterase from , have been exhaustively characterized and their crystal structures are available. Differences in substrate affinity among these proteins have been attributed to changes in their hydrophobicity. In this work, we analyzed the full catalytic mechanisms of these proteins using molecular dynamics tools, gaining insight into their mechanistic properties. In addition, we developed an protocol to predict the substrate specificity using and lipases as model enzymes and triglycerides and cholesterol esters with different fatty acid chain lengths as model substrates. The protocol was validated by comparing the results with those described in the literature. These results would be useful to perform virtual screening of substrates for enzymes of the -like family with unknown catalytic properties.
Topics: Candida; Lipase; Ophiostoma; Saccharomycetales; Sterol Esterase; Substrate Specificity
PubMed: 33555857
DOI: 10.1021/acs.jcim.0c01151 -
Daru : Journal of Faculty of Pharmacy,... Jun 2021Due to lipases' regio-selectivity and ability to catalyze different reactions such as hydrolysis, esterification, and transesterification, the enzyme is attractive in...
AIM
Due to lipases' regio-selectivity and ability to catalyze different reactions such as hydrolysis, esterification, and transesterification, the enzyme is attractive in biotransformation technology. Besides, another technology, namely enzyme immobilization, has attracted scientists/technologists' attention to employ immobilized lipase in such a field. Thus lipase of Candida rugosa was immobilized onto silica nanoparticles through adsorption. Furthermore, the immobilized biocatalyst was characterized and used to esterify ibuprofen enantioselectively.
METHODS
To characterize immobilized lipase onto silica nanoparticles scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used.
RESULTS
The catalytic properties of both immobilized and free lipases such as optima pH and temperature were not different. According to the results, the immobilized lipase on silica nanoparticles showed 45% and 96% conversion (C) and enantioselectivity (ee), respectively. In comparison to free lipase, the immobilized enzyme came with better catalytic activity.
CONCLUSION
Silica nanoparticles as one of the most promising materials for the immobilization of lipase in enantioselective esterification of ibuprofen, were introduced in this work.
Topics: Adsorption; Biocatalysis; Enzymes, Immobilized; Esterification; Hydrogen-Ion Concentration; Ibuprofen; Lipase; Nanoparticles; Palmitates; Saccharomycetales; Silicon Dioxide; Temperature
PubMed: 33528796
DOI: 10.1007/s40199-021-00388-7 -
Biomedicine & Pharmacotherapy =... Oct 2020Diet and commercially available supplements can significantly impact the gut microbial composition; however, the effects of supplements often lack scientific data...
Diet and commercially available supplements can significantly impact the gut microbial composition; however, the effects of supplements often lack scientific data demonstrating the effects on healthy and diseased individuals. Hence, it was investigated, whether a frequently used supplement in humans, Candida rugosa lipase (CRL), gets delivered active beyond the stomach in the intestinal tract of C57BL/6 J mice and its impact on the gut microbial community and environment. We showed for the first time the movement of CRL in an active state through the mouse digestive tract by determination of intestinal CRL activity and free fatty acids concentrations. The short- and long-term administration of CRL resulted in significant alterations of the gut microbiome, favoring the growth of, for instance, Verrucomicrobia but also other species associated with normal body mass index (BMI) or butyrate expression, both considered beneficial. In addition, we showed that these changes persisted after supplementation and that gut barrier integrity was unaffected by the treatment. In conclusion, CRL can be delivered in an active state beyond the stomach and supplementation altered the murine gut microbiome favoring beneficial bacterial species, which may be of relevance in humans in healthy but also potentially in disease states.
Topics: Animals; Bacteria; Gastrointestinal Microbiome; Gastrointestinal Tract; Lipase; Mice, Inbred C57BL; RNA, Ribosomal, 16S; Saccharomycetales
PubMed: 32771891
DOI: 10.1016/j.biopha.2020.110579 -
Scientific Reports Jul 2020Microbial detoxification has been proposed as a new alternative for removing toxins and pollutants. In this study, the biodetoxification activities of yeasts against...
Microbial detoxification has been proposed as a new alternative for removing toxins and pollutants. In this study, the biodetoxification activities of yeasts against aflatoxin B and zinc were evaluated by HPLC and voltammetric techniques. The strains with the best activity were also subjected to complementary assays, namely biocontrol capability and heavy-metal resistance. The results indicate that the detoxification capability is toxin- and strain-dependent and is not directly related to cell growth. Therefore, we can assume that there are some other mechanisms involved in the process, which must be studied in the future. Only 33 of the 213 strains studied were capable of removing over 50% of aflatoxin B, Rhodotrorula mucilaginosa being the best-performing species detected. As for zinc, there were 39 strains that eliminated over 50% of the heavy metal, with Diutina rugosa showing the best results. Complementary experiments were carried out on the strains with the best detoxification activity. Biocontrol tests against mycotoxigenic moulds showed that almost 50% of strains had an inhibitory effect on growth. Additionally, 53% of the strains grew in the presence of 100 mg/L of zinc. It has been proven that yeasts can be useful tools for biodetoxification, although further experiments must be carried out in order to ascertain the mechanisms involved.
Topics: Aflatoxin B1; Biodegradation, Environmental; Chromatography, High Pressure Liquid; Environmental Pollutants; Food Safety; Metals, Heavy; Pichia; Rhodotorula; Saccharomyces cerevisiae; Saccharomycetales; Wastewater; Yeasts; Zinc
PubMed: 32647290
DOI: 10.1038/s41598-020-68154-4 -
Microbial Biotechnology Mar 2020Its features as a microbial and eukaryotic organism have turned Komagataella phaffii (Pichia pastoris) into an emerging cell factory for recombinant protein production...
Its features as a microbial and eukaryotic organism have turned Komagataella phaffii (Pichia pastoris) into an emerging cell factory for recombinant protein production (RPP). As a key step of the bioprocess development, this work aimed to demonstrate the importance of tailor designing the cultivation strategy according to the production kinetics of the cell factory. For this purpose, K. phaffii clones constitutively expressing (P ) Candida rugosa lipase 1 (Crl1) with different gene dosage were used as models in continuous and fed-batch cultures. Production parameters were much greater with a multicopy clone (MCC) than with the single-copy clone (SCC). Regarding production kinetics, the specific product generation rate (q ) increased linearly with increasing specific growth rate (µ) in SCC; by contrast, q exhibited saturation in MCC. A transcriptional analysis in chemostat cultures suggested the presence of eventual post-transcriptional bottlenecks in MCC. After the strain characterization, in order to fulfil overall development of the bioprocess, the performance of both clones was also evaluated in fed-batch mode. Strikingly, different optimal strategies were determined for both models due to the different production kinetic patterns observed as a trade-off for product titre, yields and productivity. The combined effect of gene dosage and adequate µ enables rational process development with a view to optimize K. phaffii RPP bioprocesses.
Topics: Gene Dosage; Pichia; Recombinant Proteins; Saccharomycetales
PubMed: 31657146
DOI: 10.1111/1751-7915.13498 -
G3 (Bethesda, Md.) Dec 2019Infections caused by opportunistic yeast pathogens have increased over the last years. These infections can be originated by a large number of diverse yeast species of...
Infections caused by opportunistic yeast pathogens have increased over the last years. These infections can be originated by a large number of diverse yeast species of varying incidence, and with distinct clinically relevant phenotypic traits, such as different susceptibility profiles to antifungal drugs, which challenge diagnosis and treatment. (syn. ) and (syn. ) are two opportunistic rare yeast pathogens, which low incidence (< 1%) limits available clinical experience. Furthermore, these yeasts have elevated Minimum Inhibitory Concentration (MIC) levels to at least one class of antifungal agents. This makes it more difficult to manage their infections, and thus they are associated with high rates of mortality and clinical failure. With the aim of improving our knowledge on these opportunistic pathogens, we assembled and annotated their genomes. A phylogenomics approach revealed that genes specifically duplicated in each of the two species are often involved in transmembrane transport activities. These genomes and the reconstructed complete catalog of gene phylogenies and homology relationships constitute useful resources for future studies on these pathogens.
Topics: Candida; Genome Size; Genome, Fungal; Genome, Mitochondrial; Phylogeny; Sequence Analysis, DNA
PubMed: 31575637
DOI: 10.1534/g3.119.400762