Continuous enzymatic interesterification of milkfat with soybean oil produces a highly spreadable product rich in polyunsaturated fatty acids

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

Signaling via cAMP in Fungi: Interconnections with Mitogen-Activated Protein Kinase Pathways

The cAMP signal transduction pathway controls a wide variety of processes in fungi. For example, considerable progress has been made in describing the involvement of cAMP pathway components in the control of morphogenesis in Saccharomyces cerevisiae, Ustilago maydis, and Magnaporthe grisea. These morphological processes include the establishment of filamentous growth in S. cerevisiae and U. maydis, and the differentiation of an appressorial infection structure in M. grisea. The discovery that appressorium formation requires cAMP signaling provides an immediate connection to fungal virulence. This connection may have broader implications among fungal pathogens because recent work indicates that cAMP signaling controls the expression of virulence traits in the human pathogen Cryptococcus neoformans. In this fungus, cAMP also influences mating, as has been found for Schizosaccharomyces pombe and as may occur in U. maydis. Finally, cAMP and mitogen- activated protein kinase pathways appear to function coordinately to control the response of certain fungi, e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe, to environmental stress. There are clues that interconnections between these pathways may be common in the control of many fungal processes.

Screening, immobilization and utilization of whole cell biocatalysts to mediate the ethanolysis of babassu oil

The screening. biomass growth of lipase-producing fungus isolated from different sources and available at URM (University Recife Mycologia). as well as, the immobilization and utilization of the whole cells for the transesterification of babassu oil were investigated. Rhizopus oryzae (URM 3231, 4692), Mucor circinelloides (URM 4140, 4182) and Penicillium citrinum URM 4216 were considered to be good intracellular lipase producers whereas those from Mucor hiemalis URM 4144 and Mucor piriformis URM 4145 were weaker. Fungi biomass containing high lipase activities was immobilized on different biomass support particles (BSPs) and with the exception of Penicillium citrinum URM 4216 all the other fungi strains exhibited high lipase activity (20-50 Ug(-1)) when immobilized in situ using polyurethane foam particles. Transesterification activities of the immobilized whole cells were evaluated in the ethanolysis reaction with babassu oil and the highest performance was attained by M. circinelloides URM 4182 giving 83.22 +/- 3.68% ester yield in less than 96 h reaction. The biocatalyst operational stability was also assessed and an inactivation profile was found to follow the Arrhenius model, revealing values of 26 days and 2.6 x 10(-2)day(-1), for half-life and a deactivation coefficient, respectively. The purified product (biodiesel) exhibited viscosity (6.63 cSt) close to the value to attend specifications by the ASTM 06751 to be used as biofuel. Results are favorable compared with data already reported in the literature and demonstrated that M. circinelloides URM 4182 whole cells is a cheaper biocatalyst that can be used in the biodiesel synthesis. (C) 2012 Elsevier B.V. All rights reserved.

Characterization of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from corn grains of different geographic origins in Brazil

Aflatoxins can cause great economic losses and serious risks to humans and animals health. The largest aflatoxin producers belong to Aspergillus section Flavi and can occur naturally in food commodities. Studies showed that molecular tools as well as the type of sclerotia produced by the strains could be helpful for identification of Aspergillus species and could be correlated with levels of toxin production. The purpose of this work was to characterize the genetic diversity using AFLP technique, the type of sclerotia and the ability of aflatoxin production by isolated strains from corn of different origins in Brazil, and to verify whether qPCR based on aflR and aflP genes is appropriate for estimating the level of aflatoxin production. All the 75 strains were classified as A. flavus and the AFLP technique showed a wide intraspecific variability within them. Regarding sclerotia production, 34% were classified as S and 66% as L type. Among the aflatoxin-producers, 52.8% produced aflatoxin B-1, while 47.2% aflatoxins B-1 and B-2. Statistical analysis showed no correlation between sclerotia production and aflatoxigenicty, and no correlation between the phylogenetic clusters and aflatoxin production. Concerning the relative expression of aflR and aflP, Pearson's correlation test demonstrated low positive correlation between the expression of the aflR and aflP genes and the production of AFB(1) and AFB(2), but showed high positive correlation between aflR and aflP expression. In contrast to the other reference strains, A. oryzae ATCC 7282 showed no amplification of aflR and aflP. The results highlight the need for detection of reliable and reproducible markers with a high positive correlation with aflatoxin production.

Characterization of a thermostable extracellular tannase produced under submerged fermentation by Aspergillus ochraceus

Background: Tannases are enzymes that may be used in different industrial sectors as, for example, food and pharmaceutical. They are obtained mainly from microorganisms, as filamentous fungi. However, the diversity of fungi stays poorly explored for tannase production. In this article, Aspergillus ochraceus is presented as a new source of tannase with interesting features for biotechnological applications. Results: Extracellular tannase production was induced when the fungus was cultured in Khanna medium with tannic acid as carbon source. The extracellular tannase was purified 9-fold with 2% recovery and a single band corresponding to 85 kDa was observed in SDS-PAGE. The native apparent molecular mass was estimated as 112 kDa. Optima of temperature and pH were 40 degrees C and 5.0, respectively. The enzyme was fully stable from 40 degrees C to 60 degrees C during 1 hr. The activity was enhanced by Mn2+ (33-39%) and NH4+ (15%). The purified tannase hydrolyzed tannic acid and methyl gallate with Km of 0.76 mM and 0.72 mM, respectively, and Vmax of 0.92 U/mg protein and 0.68 U/mg protein, respectively. The analysis of a partial sequence of the tannase encoding gene showed an open read frame of 567 bp and a sequence of 199 amino acids were predicted. TLC analysis revealed the presence of gallic acid as a tannic acid hydrolysis product. Conclusion: The extracellular tannase produced by A. ochraceus showed distinctive characteristics such as monomeric structure and activation by Mn2+, suggesting a new kind of fungal tannases with biotechnological potential. Further, it was the first time that a partial gene sequence for A. ochraceus tannase was described.

Enantioselektive Organokatalyse in Epoxidierungen und Cyanhydrinbildungen

In der vorliegenden Arbeit mit dem Titel „Enantioselektive Organokatalyse in Epoxidierungen und Cyanhydrinbildungen“ wurde die Synthese zweier Cyclophan-carbaldimine durchgeführt. Beide Verbindungen bestehen aus einem Glykosyl-Baustein und einem Paracyclophan-Baustein, die über eine Imin-Bindung verbrückt sind. Die Cyclophan-carbaldimine wurden dann als Katalysatoren in einer Reihe von enantioselektiven Reaktionen verwendet. Enantioselektive Reaktionen bilden von zwei spiegelbildlichen Produkten stets eines im Überschuss. Zu diesen Reaktionen zählen die in dieser Arbeit durchgeführten enantioselektiven Epoxidierungen und enantioselektiven Cyanhydrinsynthesen. Die enantioselektiven Epoxidierungen waren dabei als Teil der Totalsynthesen der Naturstoffe Dasyscyphin D und Caripyrin geplant. Diese beiden Naturstoffe zeigten in biologischen Tests am Institut für Biotechnologie und Wirkstoffforschung (IBWF) in Kaiserslautern Aktivität gegen den Reisbrand-Pilz Magnaporthe grisea, der für Ernteverluste in beträchtlichem Ausmaß verantwortlich ist. Das Caripyrin selbst beinhaltet eine Epoxidstruktur. Mittels der oben angeführten Katalysatoren wurde versucht, diese Epoxidstruktur selektiv einzuführen. Dies gelang nicht, aber der Naturstoff konnte mittels Epoxidierung durch m-Chlorperbenzoesäure erstmals dargestellt werden. In mehreren biologischen Vergleichstests am IBWF Kaiserslautern zeigte auch das synthetische Caripyrin mit dem natürlichen Caripyrin vergleichbare biologische Aktivität.rnDas Dasyscyphin D an sich trägt keine Epoxidfunktion. Dennoch spielt sie auch hier eine wichtige Rolle. Innerhalb der geplanten Totalsynthese von Dasyscyphin D sollte die Farnesylseitenkette eines aromatischen Ringes selektiv epoxidiert werden. Durch einen elektrophilen Angriff an dieses Epoxid sollte im Anschluss eine Cyclisierung zum Dasyscyphin-Grundgerüst eingeleitet werden, aus dem dann Dasyscyphin D dargestellt werden sollte. Im Gegensatz zur Caripyrin-Synthese, bei der die selektive Epoxidierung nicht gelang, scheint sie in der Synthese von Dasyscyphin D sattgefunden zu haben, allerdings ist eine Isolierung des Reaktionsproduktes noch nicht gelungen. Folglich konnte das Dasyscyphin D noch nicht erfolgreich synthetisiert werden.rnDie Versuche zur enantioselektiven Cyanhydrin-Synthese waren nicht Bestandteil einer Totalsynthese. Die Cyanhydrine wurden zuerst als Racemate synthetisiert und gaschromatographisch vermessen, um auf diese Weise die exakten Retentionszeiten der einzelnen Enantiomere zu ermitteln. Anschließend wurden die Cyanhydrine dann enantioselektiv dargestellt und ebenfalls gaschromatographisch vermessen. Durch den Vergleich mit den racemischen Cyanhydrinen konnte dabei direkt aus der Reaktionslösung gemessen werden, was erforderlich war, da eine Isolierung der Cyanhydrine unter den gewählten Reaktionsbedingen nicht gelang. Aus den gaschromatographischen Messungen konnten Enantiomerenüberschüsse von bis zu 95 % ermittelt werden. Weiterhin ergaben die Messungen, dass der Arabinosyl-Katalysator im Vergleich mit dem Galactosyl-Katalysator eine geringere Enantioselektivität induziert, was vermutlich auf leichte räumliche Differenzen der beiden Katalysatoren zurückzuführen ist. Ein weiteres wichtiges Ergebnis war, dass beide Katalysatoren, wie geplant, unterschiedliche Enantiomere im Überschuss bilden. Die Glykosyl- und Paracyclophan-Bausteine der beiden Katalysatoren waren so gewählt worden, dass beide Katalysatoren pseudo-Enantiomere bilden. Auf diese Weise sollte eine Einflussnahme auf das gebildete Enantiomer durch die Wahl des entsprechenden Cyclophan-carbaldimin-Katalysators möglich gemacht werden, was, wie bereits erwähnt, gelang.rn

Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses

The rice blast fungus, Magnaporthe grisea, generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of slt2 null mutants. The mps1–1Δ mutants of M. grisea have some phenotypes in common with slt2 mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly, mps1–1Δ mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although mps1–1Δ mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that MPS1 is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δchk1 mutants is attempted. The ability of Δchk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

Polygalacturonase Gene Expression in Ripe Melon Fruit Supports a Role for Polygalacturonase in Ripening-Associated Pectin Disassembly

Ripening-associated pectin disassembly in melon is characterized by a decrease in molecular mass and an increase in the solubilization of polyuronide, modifications that in other fruit have been attributed to the activity of polygalacturonase (PG). Although it has been reported that PG activity is absent during melon fruit ripening, a mechanism for PG-independent pectin disassembly has not been positively identified. Here we provide evidence that pectin disassembly in melon (Cucumis melo) may be PG mediated. Three melon cDNA clones with significant homology to other cloned PGs were isolated from the rapidly ripening cultivar Charentais (C. melo cv Reticulatus F1 Alpha) and were expressed at high levels during fruit ripening. The expression pattern correlated temporally with an increase in pectin-degrading activity and a decrease in the molecular mass of cell wall pectins, suggesting that these genes encode functional PGs. MPG1 and MPG2 were closely related to peach fruit and tomato abscission zone PGs, and MPG3 was closely related to tomato fruit PG. MPG1, the most abundant melon PG mRNA, was expressed in Aspergillus oryzae. The culture filtrate exponentially decreased the viscosity of a pectin solution and catalyzed the linear release of reducing groups, suggesting that MPG1 encodes an endo-PG with the potential to depolymerize melon fruit cell wall pectin. Because MPG1 belongs to a group of PGs divergent from the well-characterized tomato fruit PG, this supports the involvement of a second class of PGs in fruit ripening-associated pectin disassembly.

Characterization of Recombinant Rhamnogalacturonan α-l-Rhamnopyranosyl-(1,4)-α-d-Galactopyranosyluronide Lyase from Aspergillus aculeatus1 : An Enzyme That Fragments Rhamnogalacturonan I Regions of Pectin

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) α-l-rhamnopyranosyl-(1,4)-α-d-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by 1H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an α-Δ-(4,5)-unsaturated d-galactopyranosyluronic acid at the nonreducing end and an l-rhamnopyranose at the reducing end. l-Rhamnopyranose units are substituted at C-4 with β-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31°C was 28 units mg−1. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.

Rhizopus arrhizus - a producer for simultaneous saccharification and fermentation of starch waste materials to L(+)-lactic acid

Rhizopus arrhizus, strain DAR 36017, produced L(+)-lactic acid in a simultaneous saccharification and fermentation process using starch waste effluents. Lactic acid at 19.5 - 44.3 g l(-1) with a yield of 0.85 - 0.96 g g(-1) was produced in 40 h using 20 - 60 g starch l(-1). Supplementation of nitrogen source may be unnecessary if potato or corn starch waste effluent was used as a production medium.

Biotechnological production of lactic acid integrated with potato wastewater treatment by Rhizopus arrhizus

This paper describes a feasibility study of a for lactic acid production integrated with are treatment of wastewater from an industrial starch plant. Rhizopus oryzae two strains, Rhizopus arrhizus and Rhizopus oligosporus were tested with respect to their capability to carry out simultaneous saccharification and fermentation to lactic acid using potato wastewater. Rhizopus arrhizus DAR 36017 was identified as a suitable strain that demonstrated a high capacity for starch saccharification and lactic acid synthesis. The optimal conditions, in terms of pH, temperature and starch concentration, for lactic acid production were determined. The selected fungal strain grew well in a pH range from 3.0 to 7.0. The addition of CaCO(3)10 g dm(-3) maintained the pH at 5.0-6.0 and significantly enhanced lactic acid production. Kinetic study revealed that almost complete starch saccharification and a lactic acid yield of 450g kg(-1) could be achieved in 20 h and 28 h cultivation, respectively. The maximum lactic acid production 21 g dm(-3) and mycelial biomass (1.7 g dm(-3)) were obtained at 30degreesC. Besides the multiple bioproducts, total removal of suspended solids and 90% reduction of COD were achieved in a single no-aseptic operation. (C) 2003 Society of Chemical Industry.

Fungi associated with foliar diseases of wild and cultivated rice (Oryza spp.) in northern Queensland

During surveys of wild and cultivated rice in northern Queensland in 2014 and 2015, 92 fungal isolates were obtained from plants that were afflicted by foliar diseases, including the rice blast pathogen, Pyricularia oryzae, and the brown spot pathogen, Bipolaris oryzae. Seven species of Curvularia were found, viz. Curvularia aeria, C. alcornii, C. asianensis, C. clavata, C. lunata, C. muehlenbeckiae and an undescribed species. To remove uncertainty about the identity of the host plants from which the fungi were isolated, a DNA barcoding strategy was developed using regions of the chloroplast genome. Pathogenicity tests using wild rice isolates of P. oryzae indicated that many local rice varieties are susceptible to infection.

Use of sulfuryl fluoride in the management of strongly phosphine-resistant insect pest populations in bulk grain storages in Australia

Sulfuryl fluoride (SF), an effective structural fumigant, is registered recently as Profume™ for controlling insect pests of stored grains and processed commodities. Information on its effectiveness in disinfestation of bulk grain, however, is limited. The ongoing problem with the strong level of resistance to phosphine has been addressed recently through deployment of SF as a ‘resistance breaker’ in bulk storages in Australia. This paper discusses important results on the efficacy of SF against key phosphine- resistant insect pests, lesser grain borer, Rhyzopertha dominca, red flour beetle, Tribolium castaneum, rice weevil, Sitophilus oryzae and the rusty grain beetle, Cryptolestes ferrugineus. We have established CT (g-hm3) profiles for SF against these insect pests at two temperature regimes 25 and 30°C, that showed that both temperature and exposure period (t) has significant influence on the effectiveness of SF than the concentration. Over a seven days fumigation period, CTs of 800 and 400 g-hm3 achieved complete control of all the target pests, including the most strongly phosphine - resistant species, C. ferrugineus at 25 and 30°C, respectively. Results from four industry scale field trials involving currently registered rate of SF (1500 g-hm3) over 2–14 d exposure period, confirmed its effectiveness in achieving complete control of the target pest species. The assessment of postfumigation grain samples across all the test storages indicated that the reinfestation occurs after three months. Monitoring resistance to phosphine in C. ferrugineus over a six year period (2009–2015), showed a significant reduction in resistant populations after the introduction of SF into the fumigation strategy at problematic storage sites. Overall our research concludes that SF is a good candidate to be used as a ‘resistance breaker’ where phosphine resistance is prevalent.

Status of resistance to phosphine in insect pests of stored products in Vietnam

In response to numerous reports of failures to control insect pests of stored products with phosphine in Vietnam, a national survey for resistance to this key fumigant was undertaken in 2009–2011. Data from a more limited survey undertaken by the authors in 2002 in northern Vietnam are also presented. Samples collected in the 2002 survey (Sitophilus oryzae, n=8; Tribolium castaneum, n=8) were tested using a full dose- response assay, while for the 2009–11 survey, F1 generations were tested for resistance with two discriminating dosages of phosphine to detect frequency of weak and strong resistance phenotypes. Compared with a susceptible reference strain, in 2002, resistance to phosphine was indicated in six T. castaneum samples but only two of S. oryzae. Resistance factor, however, did not exceed 2.8-fold in T. castaneum and 1.7 in S. oryzae indicating relatively low frequency and weak expression of resistance. In 2009–11 survey, 176 samples were collected from a range of food and feed storages along the supply chain and from all major regions of Vietnam (125 sites). Rhyzopertha dominica and S. oryzae were the most common species found infesting stored commodities. Resistance was detected at high frequency in all the species. Weak and strong resistance phenotype frequencies were, respectively: Cryptolestes ferrugineus (37 and 58%, n=19), R. dominica (1.5 and 97%, n=65), S. oryzae (34 and 59%, n=82) and T. castaneum (70 and 30%, n=10). Strong resistance phenotype was detected in all the major regions and all parts of the supply chain but frequency was the highest in central storages and animal feed establishments. The increase in frequency and strength of resistance to phosphine in the eight years between the two surveys has been rapid and dramatic. The survey demonstrates the threat of resistance to grain protection in Vietnam and highlights the need for training of fumigators, and the development and adoption of phosphine resistance management tactics nationally.