A new approach for Chrysoporthe cubensis cellulolytic cocktail production using solid and submerged-state fermentation

Funding Information: We thank Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq) for the financial support and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing scholarships. We would like to thank Thamires Felisberto Pereira Dutra for cooperating in the experiments execution. Publisher Copyright: © 2023, The Author(s) under exclusive licence to Associação Brasileira de Engenharia Química.The lignocellulosic material bioconversion to bioproducts has received significant attention in recent years. Cellulases and hemicellulases catalyze the hydrolysis of lignocellulosic materials into fermentable sugars that are afterward converted to bioproducts by microorganisms. Chrysoporthe cubensis grown under solid-state fermentation (SSF) has produced more effective enzymatic extracts for sugarcane bagasse saccharification than commercial cellulolytic preparations. However, the investigation of new approaches for enzyme production by this fungus is still lacking. In this work, an enzyme cocktail (SSF-SmF-cocktail) was produced by extracting enzymes of C. cubensis grown under SSF using the extract produced by the same fungus under submerged fermentation (SmF). The total cellulase (FPase), carboxymethylcellulase (CMCase), cellobiohydrolase (CBH), β-glucosidase, xylanase, β-xylosidase, β-galactosidase, α-galactosidase, and α-arabinofuranosidase activities were evaluated in crude extracts obtained from C. cubensis cultivation under SSF, SmF, and also in the SSF-SmF-cocktail. The C. cubensis protein profiles cultivated under SSF and SmF were compared by SDS-PAGE. Extract produced by C. cubensis grown under SmF presented proteins with estimated molecular weights of 10.7, 29.3, 38.6, 46.0, and 170.0 kDa, respectively, but not in that produced by this fungus under SSF. When cultivated under SSF, C. cubensis produced an extract with greater protein diversity between 13 and 51 kDa than that obtained by this fungus under SmF. The 83.0 and 95.3 kDa protein bands were present in both C. cubensis cultures. The C. cubensis SSF-SmF-cocktail presented better efficiency in glucose release after 48 h of the alkali-pretreated sugarcane bagasse hydrolysis when compared to those produced by this fungus under either SSF or SmF. This extract showed the highest xylananase/FPase rate and the second highest CMCase/FPase and b-glucosidase/FPase rates among the evaluated extracts, suggesting that these enzymes are the main determinants of this cocktail the efficiency on the alkali pretreated sugarcane bagasse sacchariffication process. These results demonstrated that the enzymes produced by C. cubensis cultivated under SSF and SmF are complementary for the alkali-pretreated sugarcane bagasse enzymatic hydrolysis, since the SSF-SmF-cocktail was more efficient than other extracts produced by this fungus and that the commercial Accellerase®. Therefore, the SSF-SmF-cocktail is a promising alternative for industrial applications. Graphical abstract: [Figure not available: see fulltext.].publishersversionpublishe

Effects of nickel and nitrogen soil fertilization on lettuce growth and urease activity

Nickel is a micronutrient involved in nitrogen metabolism and a constituent of the urease molecule. Plant growth and urease activity were evaluated in lettuce (Lactuca sativa L.) grown in soil-filled pots in a 2 x 8 factorial design with two nitrogen (N) sources and eight Ni rates, with five replications. Nitrogen was applied at 200 mg dm-3 (half the dose incorporated into the soil at seedling transplanting and half top-dressed later) using the sources NH4NO3 (AN) and CO(NH2)2 (Ur). The Ni treatments (0, 2, 4, 8, 12, 16, 24 and 32 mg dm-3) were applied as NiCl2. The shoot dry-matter yield, leaf urease activity, Ni levels in the lettuce leaves and Ni levels extracted from soil with Mehlich-3 (M-3) and DTPA were determined. In the plants supplied with AN, the shoot dry-matter yield was higher than in those supplied with Ur. There was no difference in shoot dry matter in response to soil-applied Ni. The leaf urease activity increased with Ni application, regardless of the N source. The extractions with M-3 and DTPA were efficient to evaluate Ni availability for lettuce in the Red-Yellow Latosol

Effects of nickel and nitrogen soil fertilization on lettuce growth and urease activity

Nickel is a micronutrient involved in nitrogen metabolism and a constituent of the urease molecule. Plant growth and urease activity were evaluated in lettuce (Lactuca sativa L.) grown in soil-filled pots in a 2 x 8 factorial design with two nitrogen (N) sources and eight Ni rates, with five replications. Nitrogen was applied at 200 mg dm^-3 (half the dose incorporated into the soil at seedling transplanting and half top-dressed later) using the sources NH4NO3 (AN) and CO(NH2)2 (Ur). The Ni treatments (0, 2, 4, 8, 12, 16, 24 and 32 mg dm^-3) were applied as NiCl2. The shoot dry-matter yield, leaf urease activity, Ni levels in the lettuce leaves and Ni levels extracted from soil with Mehlich-3 (M-3) and DTPA were determined. In the plants supplied with AN, the shoot dry-matter yield was higher than in those supplied with Ur. There was no difference in shoot dry matter in response to soil-applied Ni. The leaf urease activity increased with Ni application, regardless of the N source. The extractions with M-3 and DTPA were efficient to evaluate Ni availability for lettuce in the Red-Yellow Latosol.Níquel é um micronutriente envolvido no metabolismo de nitrogênio e é componente da molécula da urease. Foram avaliados o crescimento de plantas e a atividade da urease em alface (Lactuca sativa L.) cultivada em solo em vasos, em experimento fatorial 2 x 8, com duas fontes de nitrogênio (N) e oito doses de Ni, com cinco repetições. O N foi aplicado ao solo (200 mg dm^-3), incorporando-se metade no transplantio das mudas e a outra metade, mais tarde, na superfície do solo, utilizando-se nitrato de amônio (NH4NO3) e ureia [CO(NH2)2]. Os tratamentos com Ni (0, 2, 4, 8, 12, 16, 24 e 32 mg dm^-3) foram aplicados, usando-se cloreto de níquel (NiCl2). Produção de matéria seca, atividade da urease e do Ni nas folhas de alface e Ni no solo (Mehlich-3 e DTPA) foram determinados. A produção de matéria seca nas plantas supridas com NH4NO3 foi maior do que nas supridas com CO(NH2)2. Não houve diferença na produção de matéria seca das folhas em resposta à aplicação de Ni ao solo. A atividade da urease aumentou com o incremento das doses de Ni, mas não se alterou em razão da fonte de N utilizada. As extrações com Mehlich-3 e DTPA foram eficientes para avaliar a disponibilidade de Ni para alface no Latossolo Vermelho-Amarelo

Caracterização de alfa-galactosidase em embrião e cotilédones de sementes de Platymiscium pubescens Micheli, var. pubecens (tamboril-da-mata)

Sementes de Platymiscium pubescens foram colocadas para embeber em água, sendo retiradas amostras para as caracterizações bioquímica e cinética da enzima alfa-galactosidase do eixo embrionário e dos cotilédones. A atividade específica no eixo embrionário aumenta de zero até o tempo de 96 horas de embebição, estabilizando em seguida. A atividade da enzima dos cotilédones mostrou pequeno incremento durante esse mesmo tempo. A alfa-galactosidase do eixo embrionário apresentou atividade máxima no intervalo de pH de 4,5 a 6,0. Por outro lado, para a enzima proveniente dos cotilédones, a maior atividade foi detectada na faixa de 4,0 a 6,0. A temperatura de 55ºC foi a que mais estimulou as atividades da alfa-galactosidase do eixo embrionário e dos cotilédones. As enzimas do eixo embrionário e dos cotilédones mostraram-se termotolerantes, não se alcançando a meia vida na temperatura de 40ºC, no período de 1.500 minutos. A atividade da alfa-galactosidase do eixo embrionário foi inibida por melibiose, CuSO4 e SDS, enquanto a dos cotilédones foi por todos os efetores, exceto com SDS, CuSO4 e galactose que tiveram efeito neutro sobre a atividade da alfa-galactosidase dos cotilédones. Os valores de KM para as alfa-galactosidases do embrião e para o cotilédone foram 3,37 e 0,26 mM, respectivamente.Platymiscium pubescens seeds were placed to soak in water and sampled for biochemistry and kinetic characterizations of embryonic axis and the alfa-galactosidase cotyledon enzyme. The specific activity in the embryonic axis increased from zero to 96 hours of imbibition, stabilizing soon after. The activity of the cotyledon enzyme showed a small increase in the same period. The alfa-galactosidase of the embryonic axis presented its maximum activity in the interval of pH 4.5 to 6.0. On the other hand, for the cotyledon enzyme, the highest activity was detected in the interval of 4.0 to 6.0. The temperature of 55ºC was best to stimulate embryonic axis and cotyledon alfa-galactosidase acticvity. Enzymes of the embryonic axis and the cotyledons were heat tolerant, not reaching the half life at 40ºC, in 1.500 minutes. The embryonic axis alfa-galactosidase activity was inhibited by melibiose, CuSO4 and SDS, while that of cotyledons was for all the effectors, except for SDS, CuSO4 and galactose which had neutral effect. The values of KM for the embryo and for the cotyledon alfa-galactosidases were 3.37 and 0.26 mM, respectively

Removal of oligosaccharides in soybean flour and nutritional effects in rats

The objectives of this work were to establish a safe and economically viable process for the removal of raffinose oligosaccharides (RO) from soy flour and compare the effects of RO elimination from diets with regard to nutritional parameters by testing in Wistar rats. Debaryomyces hansenii UFV-1 was cultivated in suspension of defatted soy flour (1:10 w/v). An increase in α-galactosidase activity was observed in the medium, with a consequent decrease in the RO concentration. A total reduction of RO was achieved at 36 h of incubation. The diet containing soy flour free of RO presented higher digestibility, 91.28%, in relation to the diet containing soy flour with RO, 87.14%. However, the removal of the oligosaccharides from the diet did not promote a significant improvement in the values of weight gain, and other nutritional parameters tested on rats, during the experimental period of 14 days

Thermostability improvement of Orpinomyces sp. xylanase by directed evolution

The methodology of directed evolution, using the mutagenic technique of error-prone PCR has been used to improve the thermostability of enzymes. This method was applied to the endo-β-1,4-xylanase from Orpinomyces strain PC-2. The constructed library of xylanase (xynA) mutants was subjected to several screening cycles in plates with azo-xylan-agarose as substrate and four thermostable mutants (M1–M4) were selected. Homology models for these thermostable mutants were constructed to identify the location of the residues changed by error-prone PCR and to investigate the effect of these mutations on the xylanase properties. Xylanase activities of the mutants and wild type were maximal at 60 °C and in the pH range of 5–7. The mutants displayed higher thermostability than the wild type XynA, where the wild type showed a half-life at 60 °C of 7.92 min, while half-life values for M1, M2, M3 and M4 were 209, 33.2, 401 and 15.3 min, respectively. Additionally, M3 and M4 presented a good performance in more extreme pH conditions. The mutants retained their ability to hydrolyze birchwood and oat spelt xylans, which are substrates presenting different degrees of branching

Components of cell wall, enzyme activity in pedicel and susceptibility of bananas to finger drop

A major problem in post-harvest handling of bananas is the individual detachment of the fruit from the hands. This study aimed to establishing the relationship between carbohydrate concentration and enzyme activity in the pedicel region of three cultivars of bananas, resistant and susceptible to natural dropping, during post-harvest ripening, and the susceptibility of bananas to finger dropping. Cultivars ‘Terra’ (plantain, AAB group) and ‘Prata’ (banana, AAB group) triploids and the ‘Prata Graúda’ (banana, AAAB group) tetraploid were used. The experiment was distributed in split plots, with three plots (cultivars) and five subplots (peel color stages) in a completely randomized design with three replications and three fruits per sample unit. ‘Terra’ showed resistance to dropping, even though the fruit were ripe, unlike ‘Prata Graúda’, which, starting from the fifth stage (yellow fruit with green tips), exhibited high susceptibility to dropping. At all ripening stages, the ‘Terra’ had the highest dry mass levels. In turn, the ‘Prata Graúda’ always maintained the lowest levels. The ‘Terra’ showed decreasing levels of pectins during ripening, whereas starch remained high even in the ripe fruit. About the enzymes studied, the results confirmed the increased resistance of the ‘Terra’ to dropping, allowing to conclude that polygalacturonase (PG) and pectinametylesterase (PME) are the key enzymes for the solubilization of the cell wall that accompanies ripening, thus playing a critical role in inducing natural dropping. The high susceptibility of the ’Prata Graúda’ to dropping is associated with the high activity of PG and PME and the low levels of dry mass; the greater resistance of the ‘Terra’ to dropping is related to higher accumulation of dry mass and starch in the pedicel.Um dos maiores problemas na comercialização de bananas é o destacamento individual dos frutos das pencas. O presente trabalho teve como objetivo estabelecer a relação entre a concentração de carboidratos e a atividade enzimática na região do pedicelo e a suscetibilidade de bananas ao despencamento natural. Foram utilizadas as cultivares triploides ‘Terra’ (plátano, grupo AAB) e ‘Prata’ (banana, grupo AAB) e a tetraplóide ’Prata Graúda’ (banana, grupo AAAB). O experimento foi conduzido em parcelas subdivididas, com três parcelas (cultivares) e cinco subparcelas (estádios de cor de casca), em um delineamento experimental inteiramente casualizado, com três repetições e três frutos constituindo a unidade amostral. A cultivar Terra mostrou resistência ao despencamento, mesmo estando seus frutos maduros, ao contrário da ‘Prata Graúda’, que, já a partir do estádio 5 (fruto amarelo com pontas verdes), exibiu suscetibilidade à queda. Em todos os estádios de amadurecimento, a cultivar ‘Terra’ teve os maiores teores de matéria seca, apresentando diminuição dos teores de pectinas no decorrer do amadurecimento, enquanto os de amido se mantiveram altos mesmo no fruto maduro. As enzimas pectinametilesterase (PME) e poligalacturonase (PG) são as enzimas-chave na solubilização da parede celular que acompanha o amadurecimento. A alta suscetibilidade da ‘Prata Graúda’ ao despencamento está associada à elevada atividade de PG e PME e ao baixo teor de matéria seca; a maior resistência ao despencamento da cultivar Terra está relacionada com o maior acúmulo de matéria seca e amido no pedicelo

Propriedades enzimáticas da enzima ALS de Cyperus difformis e mecanismo de resistência da espécie ao herbicida pyrazosulfuron-ethyl Enzymatic properties of Cyperus difformis ALS enzyme and mechanism resistance of the species to pyrazosulfuron-ethyl herbicide

Cyperus difformis L. é uma planta daninha ocorrente em lavouras de arroz irrigado, que tem apresentado dificuldade de controle devido à resistência a herbicidas inibidores da enzima ALS. Os objetivos deste trabalho foram investigar características cinéticas da enzima ALS de biótipos de C. difformis e determinar as bases bioquímicas da resistência da espécie ao herbicida pyrazosulfuron-ethyl. Para isso, foram conduzidos experimentos em laboratório do BIOAGRO/UFV. O método utilizado baseou-se na metologia utilizada por CAREY et al. (1997) e adaptada por VARGAS et al. (1999), com algumas modificações. Foram avaliadas a concentração de substrato (piruvato) que fornece velocidade inicial igual à metade da velocidade máxima de reação (K M) e velocidade máxima de reação (Vmáx), bem como a atividade da enzima ALS na presença do inibidor (pyrazosulfuron-ethyl). Diante dos resultados, pode-se observar que a resistência de C. difformis a pyrazosulfuron-ethyl é decorrente da insensibilidade da enzima ALS ao herbicida, não acarretando, porém, prejuízo aos parâmetros cinéticos K M e Vmáx da enzima ALS.<br>Cyperus difformis L. is a weed that occurs in flooded rice, which has presented difficulty in controlling due to the resistance to ALS inhibiting herbicides. The objectives of this research were to investigate kinetic characteristics of ALS enzyme from C. difformis biotypes and to determine the biochemical bases of resistance from the species to pyrazosulfuron-ethyl herbicide. For that, experiments were conducted at the BIOAGRO/UFV laboratory. The method used was based on the methodology used by CAREY et al. (1997) and adapted by VARGAS et al. (1999), with some modifications. It was evaluated substratum concentration (pyruvate) that provides initial velocity equal to half the speed reaction (K M) and maximum velocity of reaction (Vmáx), as well the activity of the ALS enzyme in the presence of the inhibitor (pyrazosulfuron-ethyl). According to the results, it is possible to observe that C. difformis resistance to pyrazosulfuron-ethyl is due to the insensibility of the ALS enzyme to the herbicide, however without penalty to K M and Vmáx kinetic parameters of the ALS enzyme

Caracterização de alfagalactosidase e sua relação com a germinação das sementes de Caesalpinia peltophoroides (Leguminosae Caesalpinioideae)

Sementes de Caesalpinia peltophoroides (Leguminosae Caesalpinioideae) – sibipiruna – foram colocadas para embebição por 144 h, sendo retiradas amostras para análise de proteína, quantificações da atividade de alfagalactosidase e de açúcares presentes na micrópila. A germinação iniciou-se com 96 h de embebição, sem que fossem detectadas modificações na parede celular da micrópila. Nesta, observou-se maior proporção de arabinose, que mostrou tendência de aumento com o decorrer da embebição. A atividade específica da alfagalactosidase foi detectada em sementes secas, tanto no eixo embrionário quanto nos cotilédones, aumentando no primeiro a partir de 24 h de embebição. O aumento da atividade nos cotilédones foi mais lento, sendo mais acentuado a partir de 120 h de embebição. O teor de proteína decresceu continuamente no eixo embrionário a partir de 24 horas de embebição, enquanto se manteve estável nos cotilédones. A atividade da alfagalactosidase foi máxima nas temperaturas de 55 e 50 ºC para o eixo embrionário e para os cotilédones, respectivamente. O pH que mais estimulou a atividade da enzima foi na faixa de 5,5 a 6,0 para o eixo embrionário e na de 4,5 a 5,0 para os cotilédones. As alfagalactosidase do eixo embrionário e dos cotilédones foram inibidas por SDS, CuSO4, galactose e melibiose. Não houve efeito estimulante sobre a atividade da alfagalactosidase do eixo embrionário por nenhum dos efetores, enquanto o mercaptoetanol estimulou a atividade da enzima dos cotilédones. Os KM para o substrato r-NPGal para a alfagalactosidase do eixo embrionário e dos cotilédones foram de 1,74 e 2,64 mM, respectivamente.Caesalpinia peltophoroides (Leguminosae Caesalpinioideae) seeds were soaked in water for 144 hours. Samples were taken for protein analysis, quantification of alphagalactosidase activity and micropyle sugar composition. Germination began after 96 hours of imbibition, with no modifications in the micropyle cellular wall. The content of arabinose was higher in the micropyle region, with a tendency to increase during imbibition. Activity of alphagalactosidase was detected in dry seeds in the embryonic axis, as well as in the cotyledons, having increased 24 hours after imbibition. The increase of the activity in the cotyledons was observed only after 120 hours of imbibition. The protein content decreased continuously in the embryonic axis after 24 hours of imbibition, however it kept stable in cotyledons. The activity of the alphagalactosidase was raised by the temperature, being maximum at 55ºC for embryonic axis and at 50ºC for cotyledons. Enzyme activity was favored by pH ranging from 5.5 to 6.0 for the embryonic axis and 4.5 to 5.0 for cotyledons. The effectors SDS, CuSO4, galactose and melibiose inhibited enzyme activity of embryonic axis and cotyledons. Mercaptoethanol showed little stimulating effect on alphagalactosidase in cotyledons. The KM of the embryonic axis and the cotyledons were 1.74 and 2.64, respectively