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Biotechnology Letters Dec 2023The applicability of a simple and high-throughput method for quantitative characterization of biofilm formation by Candida boidinii was tested in order to evaluate the...
OBJECTIVES
The applicability of a simple and high-throughput method for quantitative characterization of biofilm formation by Candida boidinii was tested in order to evaluate the effects of exogenous tyrosol on yeast growth and biofilm formation capacity.
RESULTS
Significant concentration-, temperature and time-dependent effect of tyrosol (2-(4-hydroxyphenyl)ethanol) was demonstrated, but it differentially affected the growth and biofilm formation (characterized by crystal violet staining and XTT-reduction assay) of Candida boidinii. Testing biofilm based on metabolic activity displayed sensitively the differences in the intensity of biofilm in terms of temperature, tyrosol concentration, and exposure time. At 22 °C after 24 h none of the tyrosol concentrations had significant effect, while at 30 °C tyrosol-mediated inhibition was observed at 50 mM and 100 mM concentration. After 48 h and 72 h at 22 °C, biofilm formation was stimulated at 6.25-25 mM concentrations, meanwhile at 30 °C tyrosol decreased the biofilm metabolic activity proportionally with the concentration.
CONCLUSIONS
The research concludes that exogenous tyrosol exerts unusual effects on Candida boidinii growth and biofilm formation ability and predicts its potential application as a regulating factor of various fermentations by Candida boidinii.
Topics: Biofilms; Phenylethyl Alcohol; Saccharomycetales; Candida albicans
PubMed: 37831285
DOI: 10.1007/s10529-023-03432-5 -
Applied and Environmental Microbiology Jan 2017NAD-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of...
UNLABELLED
NAD-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10C) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10C, which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances.
IMPORTANCE
FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.
Topics: Candida; Cysteine; Disulfides; Formate Dehydrogenases; Fungal Proteins; Half-Life; Mutagenesis, Site-Directed
PubMed: 27836850
DOI: 10.1128/AEM.02624-16 -
Journal of Fungi (Basel, Switzerland) Mar 2022The study of non- yeasts in wine fermentations allows the exploration of new alternatives for the reduction of ethanol in wines. The objective of this work was to...
The study of non- yeasts in wine fermentations allows the exploration of new alternatives for the reduction of ethanol in wines. The objective of this work was to evaluate the fermentation capacity of two indigenous yeasts ( and ) in monoculture and sequential fermentations (laboratory and microvinification scale) to produce Chilean Sauvignon Blanc wine. Fermentations were monitored by the determination of ethanol, glycerol, organic acids, and residual sugars. The results indicated that at the laboratory scale for both the monoculture and sequential fermentations it was possible to reduce the ethanol concentration on 0.77% / (monoculture) and 1.5% / (sequential) for and 0.50% / (monoculture) and 0.04% / (sequential) for compared to (12.87% /). Higher glycerol concentrations were produced in monoculture than sequential fermentations (: 9.47 g/L and 10.97 g/L). For microvinifications, the monoculture and sequential fermentations with managed to reduce ethanol content by 0.17% / and 0.54% /, respectively, over the control (13.74% /). In the case of , the reduction was only observed in sequential fermentations with 0.62% /. Interestingly, grapes with higher sugar concentration resulted in wines with lees ethanol concentrations. This might be associated to the use of (13.12% /) and (13.20% /) in sequential fermentations microvinification scale.
PubMed: 35330261
DOI: 10.3390/jof8030259 -
Frontiers in Plant Science 2022Autophagy is an intracellular degradation process that is highly conserved among eukaryotes at the molecular level. The process was originally revealed in the budding... (Review)
Review
Autophagy is an intracellular degradation process that is highly conserved among eukaryotes at the molecular level. The process was originally revealed in the budding yeast, but the physiological role of autophagy in yeast cells had remained unknown as autophagy-deficient yeast mutants did now show a clear growth phenotype in laboratory conditions. In this review, we introduce the role of autophagy in the methylotrophic yeast grown on the leaf surface of . Autophagy is shown to be required for proliferation in the phyllosphere, and selective autophagic pathways such as pexophagy and cytoplasm-to-vacuole targeting (Cvt) pathway are strictly regulated during both the daily cycle and the host plant life cycle. This review describes our current understanding of the role of autophagy as a survival strategy for phyllosphere fungi. Critical functions of autophagy for pathogen invasions are also discussed.
PubMed: 35401602
DOI: 10.3389/fpls.2022.867486 -
Chembiochem : a European Journal of... Mar 2022Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P) recycling and therefore,...
Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P) recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of Rhodococcus opacus 1CP and FDH (FDH and FDH ) of Candida boidinii in different positions with His-tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20-fold when it is in the N-terminus of the fusion protein. FDH +AzoRo was the most active construct and was further characterized. Enzymatic activities of FDH +AzoRo decreased compared to parental enzymes but showed improved substrate scope - accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.
Topics: Biocatalysis; Coloring Agents; Formate Dehydrogenases; NAD; Nitroreductases
PubMed: 35080802
DOI: 10.1002/cbic.202100643 -
Foods (Basel, Switzerland) Oct 2022The purpose of this study was to explore the inoculated fermentation of cv. Kalamata natural black olives using selected strains of yeast cultures with multifunctional...
The purpose of this study was to explore the inoculated fermentation of cv. Kalamata natural black olives using selected strains of yeast cultures with multifunctional potential. For this purpose, five yeast starters belonging to (four starters) and (one starter), previously isolated from table olive fermentation of the same variety and screened for their technological characteristics and probiotic potential, were inoculated in brines at the beginning of fermentation. Microbial populations (lactic acid bacteria, yeasts, and ), pH, titratable acidity, organic acids, and ethanol were monitored during fermentation for a period of 5 months. At the same time, the survival of each starter was assessed by culture-dependent molecular identification at the beginning (0 days), middle (75 days), and final stages (150 days) of fermentation in the brines and olives (at the end of the process only). The results revealed the coexistence of yeasts and lactic acid bacteria (LAB) throughout fermentation in most processes and also the absence of after the first 20 days of brining. The population of yeasts remained 2 log cycles below LAB counts, except for in the inoculated treatment with Y28, where the yeast starter prevailed from day 60 until the end of the fermentation, as well as in the inoculated treatment with Y30, where no LAB could be detected in the brines after 38 days. At the end of the process, LAB ranged between 4.6 and 6.8 log CFU/mL, while yeasts were close to 5.0 log CFU/mL, except for the inoculated fermentation with Y27 and spontaneous fermentation (control), in which the yeast counts were close to 3.5 log CFU/mL. At the end of fermentation, the recovery percentage of Y27 was 50% in the brines and 45% in the olives. Y28 and Y34 could be recovered at 25% and 5% in the brine, respectively, whereas neither starter could be detected in the olives. For Y30, the recovery percentage was 25% in the brine and 10% in the olives. Finally, Y31 could not be detected in the brines and survived at a low percentage (10%) in the olives.
PubMed: 36230182
DOI: 10.3390/foods11193106 -
Acta Crystallographica. Section D,... Nov 2023Candida boidinii NAD-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential application in the production of biofuels and various...
Candida boidinii NAD-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential application in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of wild-type CbFDH at cryogenic and ambient temperatures, as well as that of the Val120Thr mutant at cryogenic temperature, determined at the Turkish Light Source `Turkish DeLight'. The structures reveal new hydrogen bonds between Thr120 and water molecules in the active site of the mutant CbFDH, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, these findings provide invaluable insights into future protein-engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.
Topics: Formate Dehydrogenases; Candida; Crystallography, X-Ray; Saccharomycetales
PubMed: 37860962
DOI: 10.1107/S2059798323008070 -
Biochemistry May 2016The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for...
The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.
Topics: Apoenzymes; Candida; Formate Dehydrogenases; Fungal Proteins; Kinetics; NAD; Sodium Azide
PubMed: 27100912
DOI: 10.1021/acs.biochem.6b00181 -
Bioengineered Apr 2022Owing to various undesirable health effects of sugar overconsumption, joint efforts are being made by industrial sectors and regulatory authorities to reduce sugar... (Review)
Review
Owing to various undesirable health effects of sugar overconsumption, joint efforts are being made by industrial sectors and regulatory authorities to reduce sugar consumption practices, worldwide. Artificial sweeteners are considered potential substitutes in several products, e.g., sugar alcohols (polyols), high-fructose corn syrup, powdered drink mixes, and other beverages. Nevertheless, their long-standing health effects continue to be debatable. Consequently, growing interest has been shifted in producing non-caloric sweetenersfrom renewable resources to meet consumers' dietary requirements. Except for the lysozyme protein, various sweet proteins including thaumatin, mabinlin, brazzein, monellin, miraculin, pentadin, and curculin have been identified in tropical plants. Given the high cost and challenging extortion of natural resources, producing these sweet proteins using engineered microbial hosts, such as and represents an appealing choice. Engineering techniques can be applied for large-scale biosynthesis of proteins, which can be used in biopharmaceutical, food, diagnostic, and medicine industries. Nevertheless, extensive work needs to be undertaken to address technical challenges in microbial production of sweet-tasting proteins in bulk. This review spotlights historical aspects, physicochemical properties (taste, safety, stability, solubility, and cost), and recombinant biosynthesis of sweet proteins. Moreover, future opportunities for process improvement based on metabolic engineering strategies are also discussed.
Topics: Bioprospecting; Biotechnology; Plant Proteins; Recombinant Proteins; Sweetening Agents; Taste
PubMed: 35435127
DOI: 10.1080/21655979.2022.2061147 -
Genome Announcements Jan 2016Candida boidinii and Candida sojae yeasts were isolated from energy cane bagasse and plague-insects. Both have fast xylose uptake rate and produce great amounts of...
Candida boidinii and Candida sojae yeasts were isolated from energy cane bagasse and plague-insects. Both have fast xylose uptake rate and produce great amounts of xylitol, which are interesting features for food and 2G ethanol industries. Because they lack published genomes, we have sequenced and assembled them, offering new possibilities for gene prospection.
PubMed: 26769937
DOI: 10.1128/genomeA.01551-15