Ana Paula Trovatti Uetanabaro

D-xylose-fermenting and xylanase-producing yeast species from rotting wood of two Atlantic Rainforest habitats in Brazil.

This study investigated the yeast species associated with rotting wood in Brazilian Atlantic Rainforest ecosystems focusing on the identification of D-xylose-fermenting and/or xylanase-producing species. A total of 321 yeast strains were isolated from rotting wood samples collected in two Atlantic Rainforest areas. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Schwanniomyces polymorphus, Scheffersomyces queiroziae, Barnettozyma californica, and Candida (Ogataea) boidinii were the most frequently isolated yeasts. The rarefaction curves for the yeast communities isolated in YNB-D-xylose and YNB-xylan from both areas continued to rise and did not reach an asymptote, indicating that not all yeast diversity had been recovered. Additionally, the yeast composition was variable among the samples and areas, which was confirmed by the values of the Sorensen index. Among the 69 species identified, only 12 were found in both areas sampled. Fifteen possible new species were obtained. Among them, two species (Sugiyamaella sp. 1 and Sugiyamaella xylanicola) showed the ability to ferment D-xylose into ethanol, and three species (Spencermartinsiella sp. 1, Su. xylanicola and Tremella sp.) were able to produce extracellular xylanases. Indeed, most of the xylanase-producing isolates belong to the new species Su. xylanicola, which was also positive for D-xylose fermentation. S.queiroziae and S. stipitis were the main D-xylose-fermenting yeasts identified. The results of this work showed that rotting wood collected from the Atlantic Rainforests is a huge source of yeasts, including new species, with promising biotechnological properties.

Three novel ascomycetous yeast species of the Kazachstania clade, Kazachstania saulgeensis sp. nov., Kazachstania serrabonitensis sp. nov. and Kazachstania australis sp. nov. Reassignement of Candida humilis to Kazachastania humilis f. a. comb nov and Candida pseudohumilis to Kazachstania pseudohumilis f. a. comb. nov.

Five ascosporogenous yeast strains related to the genus Kazachstania were isolated. Two strains (CLIB 1764T and CLIB 1780) were isolated from French sourdoughs; three others (UFMG-CM-Y273T, UFMG-CM-Y451 and UFMG-CM-Y452) were from rotting wood in Brazil. The sequences of the French and Brazilian strains differed by one and three substitutions, respectively, in the D1/D2 large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS). The D1/D2 LSU rRNA sequence of these strains differed by 0.5 and 0.7 % from Kazachstania exigua, but their ITS sequences diverged by 8.1 and 8.3 %, respectively, from that of the closest described species Kazachstania barnettii. Analysis of protein coding sequences of RPB1, RPB2 and EF-1α distinguished the French from the Brazilian strains, with respectively 3.3, 6 and 11.7 % substitutions. Two novel species are described to accommodate these newly isolated strains: Kazachstania saulgeensis sp. nov. (type strain CLIB 1764T=CBS 14374T) and Kazachstania serrabonitensis sp. nov. (type strain UFMG-CM-Y273T=CLIB 1783T=CBS 14236T). Further analysis of culture collections revealed a strain previously assigned to the K. exigua species, but having 3.8 % difference (22 substitutions and 2 indels) in its ITS with respect to K. exigua. Hence, we describe a new taxon, Kazachstania australis sp. nov. (type strain CLIB 162T=CBS 2141T), to accommodate this strain. Finally, Candida humilis and Candida pseudohumilis are reassigned to the genus Kazachstania as new combinations. On the basis of sequence analysis, we also propose that Candida milleri and Kazachstania humilis comb. nov. are conspecific.

Genomic analysis and D-xylose fermentation of three novel Spathaspora species: Spathaspora girioi sp. nov., Spathaspora hagerdaliae f. a., sp. nov. and Spathaspora gorwiae f. a., sp. nov.

Three novel D-xylose-fermenting yeast species of Spathaspora clade were recovered from rotting wood in regions of the Atlantic Rainforest ecosystem in Brazil. Differentiation of new species was based on analyses of the gene encoding the D1/D2 sequences of large subunit of rRNA and on 642 conserved, single-copy, orthologous genes from genome sequence assemblies from the newly described species and 15 closely-related Debaryomycetaceae/Metschnikowiaceae species. Spathaspora girioi sp. nov. produced unconjugated asci with a single elongated ascospore with curved ends; ascospore formation was not observed for the other two species. The three novel species ferment D-xylose with different efficiencies. Spathaspora hagerdaliae sp. nov. and Sp. girioi sp. nov. showed xylose reductase (XR) activity strictly dependent on NADPH, whereas Sp. gorwiae sp. nov. had XR activity that used both NADH and NADPH as co-factors. The genes that encode enzymes involved in D-xylose metabolism (XR, xylitol dehydrogenase and xylulokinase) were also identified for these novel species. The type strains are Sp. girioi sp. nov. UFMG-CM-Y302(T) (=CBS 13476), Sp. hagerdaliae f.a., sp. nov. UFMG-CM-Y303(T) (=CBS 13475) and Sp. gorwiae f.a., sp. nov. UFMG-CM-Y312(T) (=CBS 13472).

Thermostable inulinases secreted by yeast and yeast-like strains from the Brazilian semi-arid region

The use of inulinases provides an alternative to the chemical process of inulin hydrolysis to obtain fructose syrup, and can reduce processing steps, time, and costs in the food industry. The objective of this work was to screen the thermostable inulinases produced by yeast and yeast-like strains isolated from the Brazilian semi-arid region. Thermostability was studied at different temperatures (60°C, 70°C, 80°C and 90°C) and for increasing periods of time (0–50 min). Thirty-three microorganisms were tested, and 27 showed inulinase activity with specific activities ranging from 0.98 to 73.79 µmol/mg protein/min. Three strains (CCMB 300, CCMB327 and CCMB328) showed the desired combination of high specific activity and a small reduction in residual activity when submitted to heat treatment (≥60°C). Our results indicate that the inulinases produced by these three yeast strains from the Brazilian semi-arid region have great potential to be used for inulin hydrolysis in the food industry.

Thermoresistant xylanases from Trichoderma stromaticum: Application in bread making and manufacturing xylo-oligosaccharides

The enzymes Xyl1 and Xyl2 from T. stromaticum were purified and identified by mass spectrometry (MALDI-TOF/MS). Xyl1 contained three proteins with similarity to xylanase family 10, 62 and anarabinofuranosidase of the Trichoderma genus and Xyl2 contained a protein with similarity to endo-1,4-β-xylanase. High xylanase activity was found at 50°C for Xyl1 and 60°C for Xyl2 and pH 5.0 for both, retaining more than 80% of activities for one hour at 60°C and pH 5-8. Ag2+ and β-mercaptoethanol increased while SDS and EDTA inhibited the xylanase activity of both Xyl1 and Xyl2 extracts. The Km and Vmax values for purified Xyl2 were 9.6mg/mL and 28.57μmol/min/mg, respectively. In application tests, both Xyl1 and Xyl2 were effective in degrading beechwood xylan to produce xylo-oligosaccharides. In baking, adding Xyl1 increased the softness and volume of wheat bread and whole grain bread, qualities increasingly desired by consumers in this segment.

Atividade antimicrobiana de méis de cinco espécies de abelhas brasileiras sem ferrão

The antimicrobial activity of honey produced by Melipona asilvai, Melipona quadrifasciata anthidioides, Friseomelita doederleinei, Tetragonisca angustula and Plebeia sp. were investigated. The agar well diffusion assay demonstrated that all honeys had antibacterial activity against Staphylococcus aureus, but only the samples from M. quadrifasciata anthidioides and F. doederleinei inhibited the growth of Escherichia coli. In the Minimum Inhibitory Concentration determination assay, M. asilvai, M. quadrifasciata anthidioides, F. doederleinei and T. angustula honeys were more active than that from Plebeia sp. for S. aureus and E. coli. The microorganisms Pseudomonas aeruginosa and Candida albicans were resistant to the all native stingless bee honeys in both assays. Honeys were more effective against bacteria than a sugar solution, suggesting that the mechanism for bacterial growth inhibition is not only related to the osmotic effect. The results of antimicrobial activity may explain the popular medicinal use of these honeys in bacterial diseases.

Identification and characterization of a class III chitin synthase gene of Moniliophthora perniciosa, the fungus that causes witches’ broom disease of cacao

Chitin synthase (CHS) is a glucosyltransferase that converts UDP-N-acetylglucosamine into chitin, one of the main components of fungal cell wall. Class III chitin synthases act directly in the formation of the cell wall. They catalyze the conversion of the immediate precursor of chitin and are responsible for the majority of chitin synthesis in fungi. As such, they are highly specific molecular targets for drugs that can inhibit the growth and development of fungal pathogens. In this work, we have identified and characterized a chitin synthase gene of Moniliophthora perniciosa (Mopchs) by primer walking. The complete gene sequence is 3,443 bp, interrupted by 13 small introns, and comprises a cDNA with an ORF with 2,739 bp, whose terminal region was experimentally determined, encoding a protein with 913 aa that harbors all the motifs and domains typically found in class III chitin synthases. This is the first report on the characterization of a chitin synthase gene, its mature transcription product, and its putative protein in basidioma and secondary mycelium stages of M. perniciosa, a basidiomycotan fungus that causes witches’ broom disease of cacao.

Polygalacturonase secreted by yeasts from Brazilian semi-arid environments

Microbial pectinolytic enzymes are known to play a commercially important role in a number of industrial processes. The objective of this study was to investigate the extracellular polygalacturonases of yeasts isolated from Brazilian semi-arid environments. Among the 250 colonies tested, only 33 produced extracellular polygalacturonases: Aureobasidium pullulans (18 isolates), Candida boidinii (one isolate), Trichosporonoides sp. (three isolates), Kluyveromyces marxianus (one isolate), Cryptococcus liquefaciens (one isolate), Pseudozyma sp. (four isolates), and yeast-like related to fungal endophyte (five isolates). The highest activity of polygalacturonase was observed in Pseudozyma sp. CCMB 300 (14.17±0.08 µmol acid galacturonic released/min/mg protein). This study shows the potential of yeasts and yeast-like organisms isolated from Brazilian semi-arid environments to produce pectinolytic enzymes.

Chemical composition and pharmacological properties of the essential oils obtained seasonally from Lippia thymoides.

Abstract Context: Lippia thymoides Mart. & Schauer (Verbenaceae) is used in folk medicine to treat wounds, fever, bronchitis, rheumatism, headaches, and weakness. Objective: This study determinates the chemical composition of essential oils from L. thymoides, obtained at during each of the four seasons and correlates with pharmacological properties. Materials and methods: Essential oils were obtained by hydrodistillation and analyzed by gas chromatography coupled to mass spectroscopy (GC-MS). Antioxidant activity was determined by DPPH free radical scavenging and β-carotene bleaching methods. The antimicrobial assays were performed by minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) methods. Isolated rat aorta and uterus, and guinea-pig trachea were utilized to evaluate relaxant potential in pre-contracted smooth muscle. Results and discussion: Essential oils from leaves of L. thymoides had the sesquiterpene β-caryophyllene (17.22–26.27%) as the major constituent followed by borneol (4.45–7.36%), camphor (3.22–8.61%), camphene (2.64–5.66%), and germacrene D (4.72–6.18%). In vitro assays showed that these essential oils do not have antioxidant activity, have antimicrobial selectivity to Gram-positive bacteria Staphylococcus aureus (MIC = 0.004 mg/mL and MMC = 0.26–10.19 mg/mL) and Micrococcus luteus (MIC = 0.03 mg/mL and MMC = 8.43 mg/mL), relax isolated rat aorta (EC50 = 305–544 μg/mL, with endothelium; and EC50 = 150–283 μg/mL, without endothelium), and uterus (EC50 = 74–257 μg/mL), and minor potency, isolated guinea-pig trachea. Conclusions: Lippia thymoides is a source of natural products of pharmaceutical interest, being necessary additional studies to determine the substances involved in the biological activities.

Actinobacteria from Termite Mounds Show Antiviral Activity against Bovine Viral Diarrhea Virus, a Surrogate Model for Hepatitis C Virus.

Extracts from termite-associated bacteria were evaluated for in vitro antiviral activity against bovine viral diarrhea virus (BVDV). Two bacterial strains were identified as active, with percentages of inhibition (IP) equal to 98%. Both strains were subjected to functional analysis via the addition of virus and extract at different time points in cell culture; the results showed that they were effective as posttreatments. Moreover, we performed MTT colorimetric assays to identify the CC50, IC50, and SI values of these strains, and strain CDPA27 was considered the most promising. In parallel, the isolates were identified as Streptomyces through 16S rRNA gene sequencing analysis. Specifically, CDPA27 was identified as S. chartreusis. The CDPA27 extract was fractionated on a C18-E SPE cartridge, and the fractions were reevaluated. A 100% methanol fraction was identified to contain the compound(s) responsible for antiviral activity, which had an SI of 262.41. GC-MS analysis showed that this activity was likely associated with the compound(s) that had a peak retention time of 5 min. Taken together, the results of the present study provide new information for antiviral research using natural sources, demonstrate the antiviral potential of Streptomyces chartreusis compounds isolated from termite mounds against BVDV, and lay the foundation for further studies on the treatment of HCV infection.