Application of methylotrophic yeast Pichia pastoris in the field of food industry - A review

Since ancient times, the utilization of yeasts by the man has a great impact on the socio-economic development. After the advent of the technology of recombinant DNA, great advances have occurred due to the acquisition of strains of mutant yeasts in the field of applied research, and Saccharomyces cerevisiae has soon been outstanding as an interesting candidate for the expression of heterologous proteins of biotechnological interest. As the time goes by other alternative systems of expression have been shown because they have advantages over Saccharomyces cerevisiae. Among those new systems, Pichia pastoris is outstanding as methylotrophic yeast capable of growing in a culture medium containing methanol as the only source of carbon and energy. The induction of production of glycerol-3-phosphate dehydrogenase (GPD, NAD(+): oxido-redutase EC 1.1. 1.8) by Pichia pastoris was accomplished in the medium containing methanol. One of the most important key parameters in Pichia pastoris expression system is the methanol concentration. Bibliographic reviews on the Pichia pastoris production system have shown that the best culture conditions vary according to the strain used and/or kind of heterologous protein desired to be expressed. Therefore, we have sought to develop a system, involving expression of glycerol-3-phosphate dehydrogenase in the yeast Pichia pastoris, for generating sufficient quantities of the enzyme in order to asses its potential value for use in various food bioanalytical determination. Dehydrogenases have been widely used in the enzymatic assays of diverse composites of industrial interest, being enclosed among them glycerol and a number of important bioanalytical applications.

Ligand Exchange Inducing Efficient Incorporation of CisPt Derivatives into Ureasil-PPO Hybrid and Their Interactions with the Multifunctional Hybrid Network

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Controlled Cisplatin Delivery from Ureasil-PEO1900 Hybrid Matrix

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Distribution of exoantigens and a 43-kDa glycoprotein (gp43) in the yeast and mycelial forms of Paracoccidioides brasiliensis

Objective. Paracoccidioides brasiliensis antigens (strain 113) were located at ultrastructural level in both yeast and mycelial forms of the fungus. The reactivity of the sera employed was analysed. Materials and methods. Immunofluorescence and ultrastructural protein A-gold immunolabelling techniques were performed using two polyclonal antisera: one against P. brasiliensis exoantigens and the other against a 43-kDa glycoprotein (gp43). Immunoblotting assays were employed to define reactivity of these antisera with somatic and metabolic antigens of both forms of the fungus. Results. The techniques employed revealed in both yeast and mycelial forms of P. brasiliensis a similar antigenic distribution. The antigens deposits were seen within the cytoplasm, and over the cell wall of the fungus. The anti-exoantigen serum recognized several bands in both forms of the fungus. The anti-gp43 serum reacted strongly with the 43-kDa fraction and weakly with few other fractions. Conclusions. Immunocytochemical techniques suggest a protein synthesis within the cytoplasm followed by excretion through the cell wall. Similar results employing both polyclonal antisera were obtained.

Growth-promoting factors for yeast cells of Paracoccidioides brasiliensis

Low-density seedings of yeast cells of Paracoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of six P. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings of P. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2-4%, moderately or considerably promoted the growth of these P. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells of P. brasiliensis on conventional mycological agar media.

Paternity study of an interspecific natural hybrid of the genus Eucalyptus L'Hér (Myrtaceae) based on cytogenetic data

Three species of the genus Eucalyptus (E. dunni, E. grandis, E. saligna) and interspecific hybrid were studied cytogenetically. The Eucalyptus species and the hybrid showed a symmetrical karyotype with 2n=22 chromosomes, with chromosome length ranging from 0.67 to 1.39 μm. Karyotypic analysis indicated a homogenous morphology and chromosome number for the species and the hybrid studied here. Based on the karyotype asymmetry data, together with the chromosome morphology results, the hybrid presented close similarity to E. saligna, suggesting that the latter is one of the parental species involved in the production of the hybrid.

Strong selection against hybrids at a hybrid zone in the Ensatina ring species complex and its evolutionary implications

The analysis of interactions between lineages at varying levels of genetic divergence can provide insights into the process of speciation through the accumulation of incompatible mutations. Ring species, and especially the Ensatina eschscholtzii system exemplify this approach. The plethodontid salamanders E. eschscholtzii xanthoptica and E. eschscholtzii platensis hybridize in the central Sierran foothills of California. We compared the genetic structure across two transects (southern and northern Calaveras Co.), one of which was resampled over 20 years, and examined diagnostic molecular markers (eight allozyme loci and mitochondrial DNA) and a diagnostic quantitative trait (color pattern). Key results across all studies were: (1) cline centers for all markers were coincident and the zones were narrow, with width estimates of 730 m to 2000 m; (2) cline centers at the northern Calaveras transect were coincident between 1981 and 2001, demonstrating repeatability over five generations; (3) there were very few if any putative F1s, but a relatively high number of backcrossed individuals in the central portion of transects: and (4) we found substantial linkage disequilibrium in all three studies and strong heterozygote deficit both in northern Calaveras, in 2001, and southern Calaveras. Both linkage disequilibrium and heterozygote deficit showed maximum values near the center of the zones. Using estimates of cline width and dispersal, we infer strong selection against hybrids. This is sufficient to promote accumulation of differences at loci that are neutral or under divergent selection, but would still allow for introgression of adaptive alleles. The evidence for strong but incomplete isolation across this centrally located contact is consistent with theory suggesting a gradual increase in postzygotic incompatibility between allopatric populations subject to divergent selection and reinforces the value of Ensatina as a system for the study of divergence and speciation at multiple stages. © 2005 The Society for the Study of Evolution. All rights reserved.

A review on the development and properties of continuous fiber/epoxy/aluminum hybrid composites for aircraft structures

Weight reduction and improved damage tolerance characteristics were the prime drivers to develop new family of materials for the aerospace/ aeronautical industry. Aiming this objective, a new lightweight Fiber/ Metal Laminate (FML) has been developed. The combination of metal and polymer composite laminates can create a synergistic effect on many properties. The mechanical properties of FML shows improvements over the properties of both aluminum alloys and composite materials individually. Due to their excellent properties, FML are being used as fuselage skin structures of the next generation commercial aircrafts. One of the advantages of FML when compared with conventional carbon fiber/epoxy composites is the low moisture absorption. The moisture absorption in FML composites is slower when compared with polymer composites, even under the relatively harsh conditions, due to the barrier of the aluminum outer layers. Due to this favorable atmosphere, recently big companies such as EMBRAER, Aerospatiale, Boing, Airbus, and so one, starting to work with this kind of materials as an alternative to save money and to guarantee the security of their aircrafts.

Correlation of the hybrid layer thickness and resin tags length with the bond strength of a self-etching adhesive system.

The objective of this study was to measure the thickness of the hybrid layer (HLT), length of resin tags (RTL) and bond strength (BS) in the same teeth, using a self-etching adhesive system Adper Prompt L Pop to intact dentin and to analyze the correlation between HLTand RTL and their BS. Ten human molars were used for the restorative procedures and each restored tooth was sectioned in mesio-distal direction. One section was submitted to light microscopy analysis of HLT and RTL (400x). Another section was prepared and submitted to the microtensile bond test (0.5 mm/min). The fractured surfaces were analyzed using scanning electron microscopy to determine the failure pattern. Correlation between HLT and RTL with the BS data was analyzed by linear regression. The mean values of HLT, RTL and BS were 3.36 microm, 12.97 microm and 14.10 MPa, respectively. No significant relationship between BS and HLT (R2= 0.011, p>0.05) and between BS and RTL (R2= 0.038) was observed. The results suggested that there was no significant correlation between the HLT and RTL with the BS of the self-etching adhesive to dentin.

Analysis of hybrid layer thickness, resin tag length and their correlation with microtensile bond strength using a total etch adhesive to intact dentin.

The objective of this study was to evaluate the use of a two-step total etch and rinse adhesive, the correlation between the hyybrid layer thickness (HL) and bond strength (BS), and between resin tag length (RT) and bond strength in the same teeth, and also to evaluate the fracture patterns of the tested specimens. Ten human molars were used for the restorative procedure and then sectioned in two halves (mesio-distally). The materials used were Adper Single Bond 2, 3M ESPE, Ultra etch gel, Ultradent and Filtek Z250, 3M ESPE. One half were utilized to measure the HL thickness and RT length through light microscopy analysis (400x), and the other half was subject to a microtensile test to measure the BS. The fractured surfaces were analyzed by scanning electron microscopy and fracture patterns classified. The Pearson correlation test was applied (p = 0.05). The results of the analyses of each specimen then were correlated: mean HL thickness = 4.39 (0.48) microm, mean length of RT = 9.94 (1.69) microm, mean BS = 23.98 (10.24) MPa. A statistically significant correlation between HL thickness and bond strength was found (r = 0.93). The two step etch and rinse adhesive system, showed a strong correlation between HL thickness and bond strength. The most common fractures were adhesive, followed by cohesive in resin.

Influence of the hybrid layer thickness and resin tag length on microtensile bond strength.

The purpose of this study was to evaluate the correlation between the hybrid layer thickness/resin tag length and the microtensile bond strength of conventional two-step adhesive system, when applied to healthy dentinal tissue. After performing the restorative adhesive procedures and tooth extractions, ten specimens were sectioned in the mesiodistal direction. One section was used for microscopic analysis of the resin tag lengths and the hybrid layer thickness, while the other was used for the microtensile bond strength test (0.5 mm/min). The fractured surface was classified according to the fracture pattern, under a stereoscopic microscope at 40x magnification. Data obtained were submitted to analysis using one-way ANOVA and Pearson's Correlation test (alpha=0.05). The means corresponding to the hybrid layer thickness, resin tag lengths and the microtensile test were 2.68 microm, 6.43 microm and 16.23 MPa, respectively. There was no correlation between the means of the values obtained for the microtensile test, and those presented by the hybrid layer (r2=0.40, p>0.05) and resin tags (r2=0.21, p>0.05). The microtensile bond strength of the conventional two-step adhesive system Adper Single Bond 2 did not depend on the thickness of the hybrid layer and length of resin tags.

Correlation between hybrid layer thickness, resin tag length and microtensile bond strength of a self-etching adhesive system

The purpose of this study was to evaluate the correlation between the hybrid layer thickness, resin tag length and resin bond strength of a self-etching adhesive system to sound dentin tissue in vivo. After performing restorative procedures and tooth extractions, ten specimens were sectioned in a mesiodistal direction. One dental section was used for light microscope analysis, in which both the resin tag length and hybrid layer thickness were measured, while the other section was analyzed using a microtensile test (0.5 mm/min). The fractured surface of the latter section was characterized using a stereoscopic magnifying glass (40x magnifcation). The results were subject to statistical analysis using the Pearson Correlation Test (a = 0.05). The hybrid layer thickness, resin tag length and resin bond strength mean values were 2.19 microm (0.34), 4.34 microm (0.28) and 9.73 MPa (5,55), respectively. In addition, correlation tests between the resin tag length and the resin bond strength (r=0.014) and also between the hybrid layer thickness and bond strength (r=0.43), showed no statistically significant correlation. The microtensile bond strength of Adper Prompt L Pop self-etching adhesive system does not depend on hybrid layer thickness or resin tag length.

Antifungal Efficacy during Candida krusei Infection in Non-Conventional Models Correlates with the Yeast In Vitro Susceptibility Profile

The incidence of opportunistic fungal infections has increased in recent decades due to the growing proportion of immunocompromised patients in our society. Candida krusei has been described as a causative agent of disseminated fungal infections in susceptible patients. Although its prevalence remains low among yeast infections (2-5%), its intrinsic resistance to fluconazole makes this yeast important from epidemiologic aspects. Non mammalian organisms are feasible models to study fungal virulence and drug efficacy. In this work we have used the lepidopteran Galleria mellonella and the nematode Caenorhabditis elegans as models to assess antifungal efficacy during infection by C. krusei. This yeast killed G. mellonella at 25, 30 and 37°C and reduced haemocytic density. Infected larvae melanized in a dose-dependent manner. Fluconazole did not protect against C. krusei infection, in contrast to amphotericin B, voriconazole or caspofungin. However, the doses of these antifungals required to obtain larvae protection were always higher during C. krusei infection than during C. albicans infection. Similar results were found in the model host C. elegans. Our work demonstrates that non mammalian models are useful tools to investigate in vivo antifungal efficacy and virulence of C. krusei. © 2013 Scorzoni et al.

A new hybrid polyhedral cubic silsesquioxane chemically modified with 4-amino-5-(4-pyridyl)-4H-1,2,4-triazole-3-thiol (APTT) and copper hexacyanoferrate(III) for voltammetric determination of nitrite

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Yeast proteins originated from the production of brazilian bioethanol quantification and content

The purpose of this work was to determine the levels of protein and the amino acid distribution in the cell mass of yeast strains (Saccharomyces sensu stricto) originated from Brazilian bioethanol industries. The protein was analyzed with the Kjeldahl method and the amino acids, by using high-performance liquid chromatography (HPLC). The percentages of the protein found ranged from 39 to 49%. The results show that in spite of some variation in numbers between the different yeast strains, all of them presented an amino acid profile similar to the one in the literature for S. cerevisae. The amino acids that have occurred in the largest amounts were: aspartic, glutamic acids and lysine, and those in the lowest amounts were: cysteine and methionine. Although the characteristics of the feedstock used and the process conditions are determinant of the protein values obtained in dry mass, this work elucidates that the intrinsic properties of the yeast strain influence these values.