RESUMO
The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source.
Assuntos
Desidrogenases de Carboidrato , Proteínas Recombinantes , Desidrogenases de Carboidrato/metabolismo , Proteínas Recombinantes/metabolismo , Concentração de Íons de Hidrogênio , Dissacarídeos/biossíntese , Dissacarídeos/metabolismo , Temperatura , Celobiose/metabolismo , Sordariales/enzimologia , Hidrólise , Eurotiales/enzimologiaRESUMO
The transport of substrates across the cell membrane plays an essential role in nutrient assimilation by yeasts. The establishment of an efficient microbial cell factory, based on the maximum use of available carbon sources, can generate new technologies that allow the full use of lignocellulosic constituents. These technologies are of interest because they could promote the formation of added-value products with economic feasibility. In silico analyses were performed to investigate gene sequences capable of encoding xylose transporter proteins in the Candida tropicalis genome. The current study identified 11 putative transport proteins that have not yet been functionally characterized. A phylogenetic tree highlighted the potential C. tropicalis xylose-transporter proteins CtXUT1, CtXUT4, CtSTL1, CtSTL2, and CtGXT2, which were homologous to previously characterized and reported xylose transporters. Their expression was quantified through real-time qPCR at defined times, determined through a kinetic analysis of the microbial growth curve in the absence/presence of glucose supplemented with xylose as the main carbon source. The results indicated different mRNA expression levels for each gene. CtXUT1 mRNA expression was only found in the absence of glucose in the medium. Maximum CtXUT1 expression was observed in intervals of the highest xylose consumption (21 to 36 h) that corresponded to consumption rates of 1.02 and 0.82 g/L/h in the formulated media, with xylose as the only carbon source and with glucose addition. These observations indicate that CtXUT1 is an important xylose transporter in C. tropicalis. KEY POINTS: ⢠Putative xylose transporter proteins were identified in Candida tropicalis; ⢠The glucose concentration in the cultivation medium plays a key role in xylose transporter regulation; ⢠The transporter gene CtXUT1 has an important role in xylose consumption by Candida tropicalis.
Assuntos
Candida tropicalis , Xilose , Candida tropicalis/genética , Candida tropicalis/metabolismo , Carbono/metabolismo , Proteínas de Transporte/genética , Biologia Computacional , Fermentação , Expressão Gênica , Glucose/metabolismo , Cinética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Pentoses/metabolismo , Filogenia , RNA Mensageiro/metabolismo , Xilitol , Xilose/metabolismoRESUMO
Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl2. Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics. KEY POINTS: ⢠Thermophilic exo-arabinanase from family GH93 ⢠Molecular basis of arabinan depolymerization.
Assuntos
Arabinose , Glicosídeo Hidrolases , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Sordariales , Especificidade por SubstratoRESUMO
Xylanases decrease the xylan content in pretreated biomass releasing it from hemicellulose, thus improving the accessibility of cellulose for cellulases. In this work, an endo-ß-1,4-xylanase from Aspergillus fumigatus var. niveus (AFUMN-GH10) was successfully expressed. The structural analysis and biochemical characterization showed this AFUMN-GH10 does not contain a carbohydrate-binding module. The enzyme retained its activity in a pH range from 4.5 to 7.0, with an optimal temperature at 60⯰C. AFUMN-GH10 showed the highest activity in beechwood xylan. The mode of action of AFUMN-GH10 was investigated by hydrolysis of APTS-labeled xylohexaose, which resulted in xylotriose and xylobiose as the main products. AFUMN-GH10 released 27% of residual xylan from hydrothermally-pretreated corn stover and 14% of residual xylan from hydrothermally-pretreated sugarcane bagasse. The results showed that environmentally friendly pretreatment followed by enzymatic hydrolysis with AFUMN-GH10 in low concentration is a suitable method to remove part of residual and recalcitrant hemicellulose from biomass.