Immobilization and stabilization of the endo-1,4-beta-xylanase of Malbranchea pulchella for production of the xylooligosaccharides

Over the last few years, the lignocellulosic biorefinery concept has been extended beyond the application of biofuel production. Innovative and efficient technologies for lignin, cellulose, and hemicellulose fractionation allow the implementation of integrated processes for the co-production of bioenergy and higher value-added bioproducts. Among the different approaches, the use of Endo-1,4--xylanases (EC 3.2.1.8) in the hydrolysis of rich-xylan feedstocks has increased in the integrated process to produce fermentable and xylooligosaccharides (XOS). Nowadays, XOS has been preferentially used as prebiotic components in the development of new functional foods for presenting additional biological benefits such as antioxidant, inflammatory, and immunomodulatory activities. In the current work, we immobilized an endo-1,4-beta-xylanase of Malbranchea pulchella (Mpxyn10) and evaluated its potential in the production of XOS from xylan from various sources. Mpxyn10 was immobilized on agarose-activated supports (Glyoxyl-, MANAE-, GLUT- and PEI-agarose) and commercial Purolite support. Values >90% of immobilization yield were obtained on aminoactivated supports (Purolite, MANAE, and PEI-agarose) after 120 min, and the highest values of activity recovery were obtained for MANAE-MpXyn10 (137%) and Purolite-MpXyn10 (142%) derivatives. MANAE- and Purolite-MpXyn10 derivatives maintained more than 90% of their activity after 24 h of incubation at 70 °C, while the residual activity of free MpXyn10 was only 11%. MpXyn10 derivatives were also active and stable over a wide range of pH (4.0-6.0) and in the presence of furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan from various sources. Maximum values of XOS (xylobiose and xylotriose) were found for xylan beechwood at 8.1 mg mL-1, birchwood at 8.6 mg mL-1, and wheat arabinoxylan at 8.9 mg mL-1 after 3 h of reaction, at 50 °C, using Purolite-MpXyn10. This derivative was reused in various reaction cycles, maintaining more than 80% of yield XOS after 6 cycles of reaction. The results obtained in this work provide a basis for the development of applications of immobilized MpXyn10 to XOS production and other high value-added product in the lignocellulosic biorefinery field.The work was supported by the following: FAPESP (São Paulo Research Foundation, grants: 2014/50884 and 2018/07522-6; Process 2020/00081-4) and National Institute of Science and Technology of Bioethanol, INCT, CNPq (grant: 465319/2014-9) and Process 301963/2017-7.info:eu-repo/semantics/publishedVersio

Identification and characterization of novel sugar transporters potentially involved in lignocellulosic biomass degradation process in Trichoderma reesei

O fungo Trichoderma reesei apresenta uma surpreendente capacidade de produzir e secretar celulases e hemicelulases, sendo o fungo industrial mais importante na produção dessas enzimas que são utilizadas, dentre outros fins, na indústria de biocombustíveis, tal como o bioetanol. Durante o processo natural de degradação da biomassa lignocelulósica, uma série de transportadores de açúcar, com diferentes afinidades frente a um substrato, são empregados pelo fungo. A identificação e caracterização destes transportadores de açúcar pode gerar uma melhor compreensão sobre a função que estes componentes exercem no sistema de transporte de açúcares e os mecanismos moleculares nos quais estas proteínas estão envolvidas na célula. Esse trabalho tem como objetivo a identificação e caracterização de novos transportadores de açúcar potencialmente envolvidos na degradação da biomassa. Para isso, diferentes ferramentas foram empregadas. No capítulo I, é descrito a caracterização de um transportador de açúcar, Tr69957, previamente identificado por nosso grupo de pesquisa através de análises in silico de dados de RNA-seq. A deleção gênica de Tr69957 em T. reesei e análises funcionais ex vivo, através da complementação gênica do transportador em uma linhagem de Saccharomyces cerevisiae (EBY. VW4000), sugere a função deste transportador no carreamento de manose, celobiose e xilose. Além disso, a deleção do transportador afetou o perfil de expressão de genes que codificam para celulases em T. reesei. No capítulo II, uma revisão contendo resultados de pesquisa provenientes do nosso grupo: Laboratório de Biotecnologia Molecular, apresenta os principais dados sobre o envolvimento de transportadores de açúcar durante o processo de degradação da biomassa lignocelulósica. Por fim, no capítulo III, através de análises proteômicas, 14 transportadores de açúcar foram identificados durante o cultivo de T. reesei em presença de celulose microcristalina e glicerol. A identificação desses transportadores de açúcar em presença de celulose demonstra potencial função biológica durante o processo de degradação.The fungus Trichoderma reesei presents a surprising capacity to produce and secrete cellulases and hemicellulases, being the most important industrial fungus in the production of these enzymes that are used, among other purposes, in the industry of biofuels, such as bioethanol. During the natural process of degradation of lignocellulosic biomass, the fungus employs several sugar transporters with different affinities to a substrate. The identification and characterization of these sugar transporters can generate insights into the role of these components in the sugar transport system and the molecular mechanisms in which these proteins are involved in the cell. This work aims at the identification and characterization of new sugar transporters potentially involved in the degradation of biomass. For this, different tools were employed. In Chapter I, the characterization of a sugar transporter, Tr69957, previously identified by our research group through in silico RNA-seq data analysis, is described. Gene deletion of Tr69957 in T. reesei and functional ex vivo analyzes, through S. cerevisiae EBY.VW4000 strain complemented with candidate sugar transporter gene, suggests the role of this transporter in mannose, cellobiose and xylose uptake. In addition, transporter deletion affected the expression profile of cellulases encoding genes in T. reesei. In chapter II, a review containing research results from our group: Laboratory of Molecular Biotechnology presents the main data on the involvement of sugar transporters during the process of degradation of lignocellulosic biomass. Finally, in chapter III, through proteomic analyzes, 14 sugar transporters were identified during the growth of T. reesei in microcrystalline cellulose and glycerol. The identification of these sugar transporters in presence of cellulose demonstrates potential biological function during the degradation process

Evidence of cAMP involvement in cellobiohydrolase expression and secretion by Trichoderma reesei in presence of the inducer sophorose

Abstract Background The signaling second messenger cyclic AMP (cAMP) regulates many aspects of cellular function in all organisms. Previous studies have suggested a role for cAMP in the regulation of gene expression of cellulolytic enzymes in Trichoderma reesei (anamorph of Hypocrea jecorina). Methods The effects of cAMP in T. reesei were analyzed through both activity and expression of cellulase, intracellular cAMP level measurement, western blotting, indirect immunofluorescence and confocal microscopy. Results To elucidate the involvement of cAMP in the cellulase expression, we analyzed the growth of the mutant strain ∆acy1 and its parental strain QM9414 in the presence of the inducers cellulose, cellobiose, lactose, or sophorose, and the repressor glucose. Our results indicated that cAMP regulates the expression of cellulase in a carbon source-dependent manner. The expression cel7a, and cel6a genes was higher in the presence of sophorose than in the presence of cellulose, lactose, cellobiose, or glucose. Moreover, intracellular levels of cAMP were up to four times higher in the presence of sophorose compared to other carbon sources. Concomitantly, our immunofluorescence microscopy and western blot data suggest that in the presence of sophorose, cAMP may regulate secretion of cellulolytic enzymes in T. reesei. Conclusions These results allow us to better understand the role of cAMP and expand our knowledge on the signal transduction pathways involved in the regulation of cellulase expression in T. reesei. Finally, our data may help develop new strategies to improve the expression of cel7a and cel6a genes, and therefore, favor their application in several biotechnology fields

Engineering of holocellulase in biomass-degrading fungi for sustainable biofuel production

Biofuels, such as bioethanol, are a clean and sustainable form of energy and have emerged as a viable alternative to fossil fuels. Plant biomass is an important raw material for the production of clean and renewable energy. The holocellulose contained in the composition of plants may be broken down into simple sugars, such as glucose, which are fermented by yeast to produce bioethanol. The conversion of glucose polymers into fermentable sugars is accomplished by enzymes known as holocellulases, which are produced by lignocellulolytic fungi. These enzymes act synergistically for the efficient degradation of cellulose polymers, and the fine and coordinated regulation of this process is performed by transcription factors (TFs). TFs are regulatory proteins that bind to the promoter region of their target genes (CAZymes, sugar transporters, signaling proteins, other TFs, etc.) to induce or repress their transcription. This review aims to understand the main regulatory mechanisms involved in plant biomass degradation by the most studied lignocellulolytic fungi Trichoderma sp., Aspergillus sp., Penicillium sp., and Neurospora crassa. In this context, the most studied TFs related to holocellulose degradation and genetic modification of TFs or promoters as a valuable tool to improve enzyme production for biotechnological purposes have been discussed. This review enables the expansion of knowledge on the regulation of the cellulolytic system of filamentous fungi and the application of this knowledge to the improvement of numerous bioproducts. Engineering TFs and promoters may yield more efficient strains that may be active in plant biomass hydrolysis. In this way, the technological processes for obtaining ethanol from lignocellulose may become more commercially viable

Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa.

Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0), neutral (pH 7.0), and alkaline (pH 10.0) medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry

Spinal cord injury: epidemiologycal study of 386 cases with emphasis on those patients admitted more than four hours after the trauma Trauma raquimedular: estudo epidemiológico de 386 casos com ênfase para aqueles pacientes admitidos após quatro horas do trauma

We studied 386 cases of spinal cord injury to analyze the follow up of the patients admitted most of the time more than four hours, the majority of the injuries happening far from the attending health service and first specialized care received long after the accident. This is a clinical study based on data collected during hospitalization of the patients, operated or not, in a Brazilian public health service. The lesion mainly seen was fracture and dislocation, isolated or on multiple levels, and the most important clinical complications were due to respiratory failure and hypotension, especially because 73.8% were from outside and they were admitted more then four hours after the trauma. The mortality rate was 11.9%, but just 2.1% had undergone a surgery. The complications resulted in major risk of death when the trauma was at the cervical level and the patients were over 50 years old, especially when admitted more than four hours after the trauma. We emphasize the importance of the first health care concerning the clinical treatment, aiming to reduce the mortality rate.Estudamos 386 casos de trauma raquimedular, observando a evolução dos pacientes admitidos na maioria das vezes após quatro horas do trauma, a maioria procedente de lugar distante do serviço de saúde. Trata-se de um estudo clínico baseado em dados coletados durante a hospitalização, de pacientes operados ou não, em serviço de saúde pública no Brasil. Na maioria das vezes a lesão principal foi fratura-luxação, isolada ou em múltiplos níveis, e as complicações clínicas mais importantes foram devido à insuficiência respiratória e hipotensão, especialmente porque 73.8% dos casos eram de lugares distantes, tendo sido admitidos mais de quatro horas após o trauma. A taxa de mortalidade foi 11,9%, sendo 2,1% em pós-operatório. As complicações resultaram em maior risco de morte quando o trauma foi ao nível cervical, idade acima de 50 anos, especialmente aqueles admitidos com mais de quatro horas. Enfatizamos a importância do primeiro atendimento na admissão, sobretudo para as alterações respiratórias e hemodinâmicas, objetivando reduzir a mortalidade

Schematic representation of putative PAC-3 binding sites (5′ -BGCCVAGV-3′).

<p>The analysis was performed using the region 1.0-kbp upstream of cellulolytic and xylanolytic genes. The position of the motifs is relative to the translation initiation codon (ATG). B = C or G or T; V = A or C or G, according to International Union of Pure and Applied Chemistry (IUPAC) norms.</p

PAC-3 influences holocellulase activity in <i>N</i>. <i>crassa</i>.

<p>Total cellulolytic (FPase) (A), endoglucanase (CMCase) (B), and xylanolytic activities (C) were assayed in the supernatant of the strains 74A, Δ<i>mus-52</i>, and Δ<i>pac-3</i> after their cultivation for eight days on sugarcane bagasse. Values show the mean of three replicates. The error bar indicates the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.</p