Mitotic gene exchanges in the arg4 Locus of Saccharomyces cerevisiae

Três novos alelos para o Locus arg4 (biossíntese de arginina) de Saccharomyces cerevisiae foram isolados, objetivando-se analisá-los quanto aos eventos de recombinação mitótica. Através de cruzamentos os novos alelos foram dispostos em configuração trans resultando em três diplóides heteroalélicos; IG 100, portador dos alelos arg4-66 e arg4-93, IG 200, portador de arg4-93 e arg4- 80 e IG 300, portador de arg4-66 e arg4-80. Os alelos arg4-80 e arg4-93 foram também utilizados juntamente com o alelo arg4-41, de posição conhecida no mapa de complementação, para a construção de dois outros diplóides, o IG 400 (arg4-80/ arg4-41) e IG 500 (arg4-93 / arg4-41). A detecção de recombinação intragênica nos pares heteroalélicos foi possível através da disposição das células auxotróficas em meio sem arginina que permitiu evidenciar os revertentes para prototrofia, que foram interpretados como conseqüência de conversão gênica mitótica restaurando alelos funcionais. Esta metodologia possibilitou observar que ocorre reversão nos pares heteroalélicos, em IG 100 e IG 200, com frequência na ordem de 10-7 prototróficos por células viáveis, enquanto que nos pares IG 300, IG 400 e IG 500, as freqüências de reversão estão na ordem de 10-5 prototróficos por células viáveis. Doses subletais de luz ultravioleta mostraram-se indutoras de recombinação mitótica, nos segmentos vizinhos ou compreendidos pelos pares heteroalélicos pertencentes aos cinco diplóides estudados. As curvas obtidas com IG 100 e IG 2001 representando as cinéticas de indução de revertentes por ultravioleta,foram não lineares contrariando os híbridos IG 300, IG 400 e IG 500 que mostraram-se lineares. Este fato coloca os pares de alelos presentes em IG 300, IG 400 e IG 500 numa situação de destaque pois as curvas lineares, não são características com a luz ultravioleta. A observação destes tipos de cinéticas pode sugerir a existência de lesões de natureza diferente provocadas direta ou indiretamente por este agente físico, ou a ação de distintos mecanismos de reparo recombinante para estas lesões, ou mesmo a existência de ambas as situações.Three new alleles in the arg4 locus (arginine biosynthesis) of Saccharomyces cerevisiae were isolated in order to study intragenic mitotic recombination events. The new alleles were paired resulting in three heteroallelic diploids IG 100 (arg4-66 and arg4-93 alleles) IG 200 (arg4-93 and arg4-80 alleles) and IG 300 (arg4-66 and arg4-80. The arg4-8C and arg4-93 alleles were also paired with the arg4-41 allele, with known position in the complementation map, for the construction of others two diploids the IG 400 (arg4-80 / arg4-41) and IG 500 (arg4-93 / arg4-41). The detectation of intragenic recombination was possible by prototrophs recovery after plating of auxotrophic cells in arginine less media. The prototrofics revertents were understood as a result of restauration of the functional alleles by occurrence of mitotic gene conversion. This method made possible to observe that the heteroallelic pairs in diploids IG 100 and IG 200 reverted in 10-7 frequencies order (prototrofics / viable cells) and IG 300, IG 400 and IG 500 reverted in the 10-5 order (prototrofics / viable cells). Ultraviolet light in sublethal doses induced mitotic recombination in neighbour segments or comprise the heteroallelic pairs in the five diploids studied. The curves represent the ultraviolet induced revertents kinetics in IG 100 and IG 200 of non linear order whereas IG 300, IG 400 and IG 500 showed linear kinetics. This last finding reveals the allelic pair in IG 300, IG 400 and IG 500 in important situation in the view of the linear curves, which are not considers characteristic for UV light used. The observation of two kinetic tipes suggests the existence of different tipes of lesion induced direct or indirecty by this fisic agente or possible the action or distinct mechanisms of recombinant repair for this lesion or even the existence of both the situations

Morphogenesis and Production of Enzymes by Penicillium echinulatum in Response to Different Carbon Sources

The effect of different carbon sources on morphology and cellulase and xylanase production of Penicillium echinulatum was evaluated in this work. Among the six carbon sources studied, cellulose and sugar cane bagasse were the most suitable for the production of filter paper activity, endoglucanases, xylanases, and β-glucosidases. However, sucrose and glucose showed β-glucosidase activities similar to those obtained with the insoluble sources. The polyacrylamide gels proved the enzymatic activity, since different standards bands were detected in the media mentioned above. Regarding morphology, it was observed that the mycelium in a dispersed form provided the greatest enzymatic activity, possibly due to greater interaction between the substrate and hyphae. These data are important in understanding the physiology of fungi and could contribute to obtaining enzyme with potential application in the technology of second generation ethanol

Cellulase and Xylanase Production by Penicillium echinulatum in Submerged Media Containing Cellulose Amended with Sorbitol

The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v) sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v) soy bran; 0.1% (w/v) wheat bran; and a solution of salts. The highest filter paper activity (FPA) ( IU·mL−1) was obtained on the seventh day in the medium containing 0.5% (w/v) sorbitol and 0.5% (w/v) cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day ( IU·mL−1) in the medium containing 0.75% (w/v) sorbitol and 0.75% (w/v) cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v) sorbitol and 0.25% (w/v) cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems

A sustainable approach of enzymatic grafting on Eucalyptus globulus wood by laccase from the newly isolated white-rot basidiomycete Marasmiellus palmivorus VE111

22 p.-5 fig.-1 graph. abst.Enzymatic grafting on eucalyptus wood with laccase from the basidiomycete Marasmiellus palmivorus VE111, recently isolated in Southern Brazil, was investigated. Three phenol and amine hydrophobic substrates and two mediator compounds were used to verify the potential of the enzyme for oxidative grafting. Laccase-assisted grafting of octyl and lauryl gallate was effectively achieved, presenting better results with mediator addition. In the water contact angle tests, the water drop kept 60% and 37% of its initial angle, with octyl gallate and lauryl gallate, respectively. Consequently, the enzymatic grafting of these compounds allowed an increase in the hydrophobicity of eucalyptus veneers (up to 10% and 5%, respectively), which is an interesting feature for the building and furniture industry to improve the durability and dimensional stability of wood. The results obtained in this work demonstrate the possibility of an environmentally friendly pathway to help solve a major challenge in wooden construction. In addition, the promising results of this work potentiate the use of a novel laccase, manufactured on-site, to be applied in sustainable processes in wood and furniture industries and other applications where hydrophobization of materials is desirable.University of Caxias do Sul (UCS), Coordination Of Improvement of Higher Education Personnel (CAPES) (Process 3255/2013) , Research Support Foundation of the State of Rio Grande do Sul (Process 16/2551-0000 246-0), Brazilian Agricultural Research Corporation (EMBRAPA) (34/2014), National Council for Scientific and Technological Development (CNPq) (Process 201925/2018-4), MINECO/FEDER (BIO2015-68387-R), Community of Madrid (S2018/EMT-4459), Xunta de Galicia (ED431C 2017/47).Peer reviewe

Lignin degradation and detoxification of eucalyptus wastes by on-site manufacturing fungal enzymes to enhance second-generation ethanol yield

12 páginas.- 5 figuras.- 4 tablas.- 103 referencias.- Supplementary data to this article can be found online at https://doi.org/10.1016/j.apenergy.2020.114493.0.00.51.01.52.0Novel laccases have promising and valuable applications in biorefineries. This investigation documents, for the first time, the potential of depolymerising and repolymerising lignin by the secretome, rich in laccases, from a newly isolated white-rot basidiomycete Marasmiellus palmivorus VE111, for further saccharification and ethanolic fermentation steps. Proteomic analyses of the secretome of M. palmivorus show that laccases are the most predominant enzyme released by this fungus. The whole crude enzymatic broth is used for the delignification of lignin in Eucalyptus globulus wood, with the aim of enhancing the saccharification by cellulolytic and xylanolytic enzymes from Penicillium echinulatum S1M29. In addition, two different strategies, namely, laccase treatment before and after enzymatic hydrolysis, are employed to detoxify steam-exploded E. globulus wood. The objective is to increase the fermentative performance by removing substances formed during the feedstock pretreatment that can inhibit microbial fermentation. The E. globulus wood delignification results in a 31% decrease in the lignin content and a 10% increase in the glucose yield after hydrolysis. An important finding of the present work is the successful wood delignification in the absence of laccase mediators. This laccase-rich preparation also demonstrates its potential in removing the phenolic inhibitors present in steam-exploded E. globulus wood, increasing the ethanol yield by an additional 10%. Furthermore, it is important to highlight that these findings are achieved in the absence of commercial enzymes, making M. palmivorus laccases a potential candidate not only for the production of biofuels but also for the generation of lignin-derived aromatic compounds for different applications in the biotechnology industry. © 2020 Elsevier LtdFunding from University of Caxias do Sul (UCS) Coordination of Improvement of Higher Education Personnel (CAPES) (Process 3255/2013), Research Support Foundation of the State of Rio Grande do Sul (Process 16/2551-0000 246-0), Brazilian Agricultural Research Corporation (EMBRAPA) (34/2014), National Council for Scientific and Technological Development (CNPq) (Process 201925/2018-4), the Biobased Industries Joint Undertaking (www.bbi-europe.eu) project EnzOx2 (H2020-BBI-PPP-2015-2-720297, www.enzox2.eu), the projects RETOPROSOST-2-CM [S2018/EMT-4459] financed by the Comunidad de Madrid, HIDROFUN [BIO2015-68387-R], and GLYSUS [RTI2018-093683-B-I00] from MINECO/FEDER, and the CSIC Project (201740E07)Peer reviewe