Gabriela Piccolo Maitan-Alfenas

The influence of Aspergillus niger transcription factors AraR and XlnR in the gene expression during growth in D-xylose, L-arabinose and steam-exploded sugarcane bagasse.

The interest in the conversion of plant biomass to renewable fuels such as bioethanol has led to an increased investigation into the processes regulating biomass saccharification. The filamentous fungus Aspergillus niger is an important microorganism capable of producing a wide variety of plant biomass degrading enzymes. In A. niger the transcriptional activator XlnR and its close homolog, AraR, controls the main (hemi-)cellulolytic system responsible for plant polysaccharide degradation. Sugarcane is used worldwide as a feedstock for sugar and ethanol production, while the lignocellulosic residual bagasse can be used in different industrial applications, including ethanol production. The use of pentose sugars from hemicelluloses represents an opportunity to further increase production efficiencies. In the present study, we describe a global gene expression analysis of A. niger XlnR- and AraR-deficient mutant strains, grown on a D-xylose/L-arabinose monosaccharide mixture and steam-exploded sugarcane bagasse. Different gene sets of CAZy enzymes and sugar transporters were shown to be individually or dually regulated by XlnR and AraR, with XlnR appearing to be the major regulator on complex polysaccharides. Our study contributes to understanding of the complex regulatory mechanisms responsible for plant polysaccharide-degrading gene expression, and opens new possibilities for the engineering of fungi able to produce more efficient enzymatic cocktails to be used in biofuel production.

Production and application of an enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum with potential for hydrolysis of sugarcane bagasse

Blending of the enzyme extracts produced by different fungi can result in favorable synergetic enhancement of the enzyme blend with regards to the main cellulase activities, as well as the inclusion of accessory enzymes that may not be as abundant in enzyme extracts produced by predominantly cellulase producing fungi. The Chrysoporthe cubensis:Penicillium pinophilum 50:50 (v/v) blend produced herein presented good synergy, especially for FPase and endoglucanase activities which were 76% and 48% greater than theoretical, respectively. This enzyme blend was applied to sugarcane bagasse previously submitted to a simple alkali pretreatment. Glucan hydrolysis efficiency reached an excess of 60% and xylan conversion exceeded 90%. Increasing the hydrolysis temperature from 45 to 50°C also resulted in a 16-20% increase in conversion of both glucan and xylan fractions. The blended enzyme extract obtained therefore showed great potential for application in the lignocellulose hydrolysis process.

Characteristics of free endoglucanase and glycosidases multienzyme complex from Fusarium verticillioides

A novel multienzyme complex, E1C, and a free endoglucanase, E2 (GH5), from Fusarium verticillioides were purified. The E1C contained two endoglucanases (GH6 and GH10), one cellobiohydrolase (GH7) and one xylanase (GH10). Maximum activity was observed at 80 °C for both enzymes and they were thermostable at 50 and 60 °C. The activation energies for E1C and E2 were 21.3 and 27.5 kJ/mol, respectively. The KM for E1C was 10.25 g/L while for E2 was 6.58 g/L. Both E1C and E2 were activated by Mn(2+) and CoCl2 while they were inhibited by SDS, CuSO4, FeCl3, AgNO4, ZnSO4 and HgCl2. E1C and E2 presented endo-β-1,3-1,4-glucanase activity. E1C presented crescent activity towards cellopentaose, cellotetraose and cellotriose. E2 hydrolyzed the substrates cellopentaose, cellotetraose and cellotriose with the same efficiency. E1C showed a higher stability and a better hydrolysis performance than E2, suggesting advantages resulting from the physical interaction between proteins.

The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails: A comparative study.

Biomass enzymatic hydrolysis depends on the pretreatment methods employed, the composition of initial feedstock and the enzyme cocktail used to release sugars for subsequent fermentation into ethanol. In this study, sugarcane bagasse was pretreated with 1% H2SO4 and 1% NaOH and the biomass saccharification was performed with 8% solids loading using 10 FPase units/g of bagasse of the enzymatic extract from Chrysoporthe cubensis and three commercial cocktails for a comparative study. Overall, the best glucose and xylose release was obtained from alkaline pretreated sugarcane bagasse. The C. cubensis extract promoted higher release of glucose (5.32 g/L) and xylose (9.00 g/L) than the commercial mixtures. Moreover, the C. cubensis extract presented high specific enzyme activities when compared to commercial cocktails mainly concerning to endoglucanase (331.84 U/mg of protein), β-glucosidase (29.48 U/mg of protein), β-xylosidase (2.95 U/mg of protein), pectinase (127.46 U/mg of protein) and laccase (2.49 U/mg of protein).

Hydrolysis of soybean isoflavones by Debaryomyces hansenii UFV-1 immobilised cells and free β-glucosidase

An intracellular β-glucosidase from Debaryomyceshansenii UFV-1 was produced in an YP medium with cellobiose as the carbon source. This enzyme was purified, characterised and presented a Mr of 65.15kDa. Yeast cells containing the intracellular β-glucosidase were immobilised in calcium alginate. The free β-glucosidase and immobilised cells containing the enzyme presented optima values of pH and temperature of 6.0 and 45°C and 5.5 and 50°C, respectively. The free enzyme maintained 62% and 47% of its original activity after 90days at 4°C and after 15days at room temperature, respectively. The immobilisation process resulted in higher enzyme thermostability at 45 and 50°C. Soy molasses treatment with the free enzyme and the immobilised cells containing β-glucosidase, for 2h at 40°C, promoted efficient hydrolysis of isoflavone glicosides to their aglycon forms. The results suggest that this enzyme could be used in the food industry, in the free or immobilised forms, for a safe and efficient process to hydrolyse isoflavone glycosides in soy molasses.

Characterization and biotechnological application of recombinant xylanases from Aspergillus nidulans

Two xylanases from Aspergillus nidulans, XlnB and XlnC, were expressed in Pichia pastoris, purified and characterized. XlnB and XlnC achieved maximal activities at 60°C and pH 7.5 and at 50°C and pH 6.0, respectively. XlnB showed to be very thermostable by maintaining 50% of its original activity after 49h incubated at 50°C. XlnB had its highest activity against wheat arabinoxylan while XlnC had the best activity against beechwood xylan. Both enzymes were completely inhibited by SDS and HgCl2. Xylotriose at 1mg/ml also totally inibited XlnB activity. TLC analysis showed that the main product of beechwood xylan hydrolysis by XlnB and XlnC was xylotetraose. An additive effect was shown between XlnB and XlnC and the xylanases of two tested commercial cocktails. Sugarcane bagasse saccharification results showed that these two commercial enzymatic cocktails were able to release more glucose and xylose after supplementation with XlnB and XlnC.

Cooperation of Aspergillus nidulans enzymes increases plant polysaccharide saccharification

Efficient polysaccharide degradation depends on interaction between enzymes acting on the main chain and the side chains. Previous studies demonstrated cooperation between several enzymes, but not all enzyme combinations have been explored. A better understanding of enzyme cooperation would enable the design of better enzyme mixtures, optimally profiting from synergistic effects. In this study, we analyzed the cooperation of several enzymes involved in the degradation of xylan, glucan, xyloglucan and crude plant biomass from Aspergillus nidulans by single and combined incubations with their polymeric substrate. Positive effects were observed between most enzymes, although not always to the same extent. Moreover, the tailor made cocktails formulated in this study resulted in efficient release of glucose from plant biomass. This study also serves as an example for the complex cooperation that occurs between enzymes in plant biomass saccharification and how expression in easily-accessible hosts, such as Pichia pastoris, can help in revealing these effects.

Expressão de Xilanases de Aspergillus nidulans em Pichia pastoris: Purificação, Caracterização e Aplicação na Hidrólise do Bagaço de Cana-de-Açúcar

O bagaco de cana-de-acucar e o residuo agricola mais abundante no Brasil e apresenta elevado potencial para ser utilizado na producao de etanol de segunda geracao. Entretanto, a grande complexidade da biomassa lignocelulosica requer diferentes enzimas para uma hidrolise eficiente. Neste trabalho, foi realizada a purificacao, a caracterizacao bioquimica, a caracterizacao cinetica e a aplicacao na hidrolise do bagaco de cana-de-acucar de duas xilanases de Aspergillus nidulans clonadas em Pichia pastoris: AN1818.2 e AN3613.2. A inducao da expressao dos genes das xilanases foi realizada em meio BMMY contendo metanol, o qual induz a ativacao do promotor AOX1, envolvido no metabolismo deste alcool. O sobrenadante do cultivo constitui-se no extrato bruto, o qual foi submetido a cromatografia de afinidade em coluna His Trap HP e, em seguida, a cromatografia de exclusao molecular em coluna Superdex 200. A temperatura otima de atividade para a xilanase AN1818.2 foi 60 oC, ja para a xilanase AN3613.2 foi 50 oC. Ambas as enzimas mostraram-se estaveis quando pre-incubadas a 50 oC e pouco estaveis quando pre-incubadas a 60 oC. A xilanase AN1818.2 apresentou um pH otimo de atividade de 7,5 e a xilanase AN3613.2 apresentou melhor atividade em pH 6,0. Ambas as xilanases foram altamente estaveis quando pre-incubadas durante uma hora em tampoes na faixa de pH de 2,5 a 13 e retornadas ao pH 5,0. As enzimas AN1818.2 e AN3613.2 foram fortemente inibidas por HgCl2, SDS, CuSO4 e xilotriose. A xilanase AN1818.2 hidrolisou melhor o substrato arabinoxilana de trigo e a xilanase AN3613.2 o substrato xilana beechwood. O valor de Km utilizando xilana beechwood como substrato foi de 1,66 e 4,23 mg/mL para as xilanases AN1818.2 e AN3613.2, respectivamente. A hidrolise do bagaco de cana-de-acucar com o coquetel Multifect CL Genencor® foi mais eficiente quando suplementada com a xilanase AN1818.2 em combinacao com a xilanase AN3613.2. Ja a hidrolise do bagaco de cana-de-acucar com o coquetel Accellerase 1500 Genencor® foi mais eficiente quando suplementada apenas com a xilanase AN3613.2.