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京都大学 / 新制・課程博士 / 博士(農学) / 甲第23238号 / 農博第2445号 / 新制||農||1083(附属図書館) / 学位論文||R3||N5328(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 渡邊 隆司, 教授 植田 充美, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Self-assembly of polysaccharide derivatives at liquid/solid interfaces was studied by surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Carboxymethyl cellulose (CMC) adsorption onto cellulose surfaces from aqueous solutions was enhanced by electrolytes, especially by divalent cations. A combination of SPR and QCM-D results showed that CMC formed highly hydrated layers on cellulose surfaces (90 to 95% water by mass). Voigt-based viscoelastic modeling of the QCM-D data was consistent with the existence of highly hydrated CMC layers with relatively low shear viscosities of ~ 10-3 N·s·m-2 and elastic shear moduli of ~ 105 N·m-2. Adsorption of pullulan 3-methoxycinnamates (P3MC) and pullulan 4-chlorocinnamates (P4CC) with different degrees of cinnamate substitution (DSCinn) onto cellulose, cellulose acetate propionate (CAP), poly(L-lactic acid) (PLLA), and methyl-terminated self-assembled monolayer (SAM-CH3) surfaces was also studied by SPR and QCM-D. Hydrophobic cinnamate groups promoted the adsorption of pullulan onto all surfaces and the adsorption onto hydrophobic surfaces was significantly greater than onto hydrophilic surfaces. SPR and QCM-D results showed that P3MC and P4CC also formed highly hydrated layers (70 to 90% water by mass) with low shear viscosities and elastic shear moduli. Finally, cellulose adsorption and activity on pullulan cinnamate (PC) and cellulose blend films were studied via QCM-D and in situ atomic force microscopy (AFM). The hydrophobicity of PC surfaces was controlled by adjusting the degree of cinnamate substitution per anhydroglucose unit (DSCinn). It was found that cellulase showed weak adsorption onto low DSCinn PC surfaces, whereas cellulase adsorbed strongly onto high DSCinn PC surfaces, a clear indication of the role surface hydrophobicity played on enzyme adsorption. Moreover, cellulase catalyzed hydrolysis of cellulose/PC and cellulose/polystyrene (PS) blend surfaces was studied. The QCM-D results showed that the cellulase hydrolysis rate on cellulose in cellulose/PC blend surfaces decreased with increasing DSCinn. AFM images revealed smooth surfaces for cellulose/PC (DSCinn = 0.3) blend surfaces and laterally phase separated morphologies for cellulose/PC (DSCinn ≥ 0.7) blend surfaces. The combination of QCM-D and AFM measurements indicated that cellulase catalyzed hydrolysis was strongly affected by surface morphology. The cellulase hydrolysis activity on cellulose in cellulose/PS blend surfaces was similar with cellulose/PC blend surfaces (DSCinn ≥ 0.7). These studies showed self-assembly of macromolecules could be a promising strategy to modify material surfaces and provided further fundamental understanding of adsorption phenomena and bioactivity of macromolecules at liquid/solid interfaces. / Ph. D.

Adsorption of xyloglucan (XG) onto thin desulfated nanocrystalline cellulose (DNC) films was studied by surface plasmon resonance spectroscopy (SPR), quartz crystal microbalance with dissipation monitoring (QCM-D), and atomic force microscopy (AFM) measurements. These studies were compared to adsorption studies of XG onto thin sulfated nanocrystalline cellulose (SNC) films and regenerated cellulose (RC) films performed by others. Collectively, these studies show the accessible surface area is the key factor for the differences in surface concentrations observed for XG adsorbed onto the three cellulose surfaces. XG penetrated into the porous nanocrystalline cellulose films. In contrast, XG was confined to the surfaces of the smooth, non-porous RC films. Surprisingly surface charge and cellulose morphology played a limited role on XG adsorption. The effect of the non-ionic surfactant Tween 80 on the adsorption of cellulase onto alkane thiol self-assembled monolayers (SAMs) on gold was also studied. Methyl (-CH3), hydroxyl (-OH) and carboxyl (-COOH) terminated SAMs were prepared. Adsorption of cellulase onto untreated and Tween 80-treated SAMs were monitored by SPR, QCM-D and AFM. The results indicated cellulase adsorption onto SAM-CH3 and SAM-COOH were driven by strong hydrophobic and electrostatic interactions, however, hydrogen bonding between cellulase and SAM-OH was weak. Tween 80 effectively hindered the adsorption of cellulase onto hydrophobic SAM-CH3 substrates. In contrast, it had almost no effect on the adsorption of cellulase onto SAM-OH and SAM-COOH substrates because of its reversible adsorption on these substrates. / Master of Science

Dissertation (PhD)--Stellenbosch University, 2007. / PCT patent registered: https://www.google.com/patents/WO2009034414A1?cl=en&dq=pct/ib2007/004098&hl=en&sa=X&ei=b7AxUsSZK4jB0gWi14HgCQ&ved=0CEkQ6AEwAg USA: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en / USA patent registered: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en / ENGLISH ABSTRACT: The conversion of cellulosic biomass into fuels and chemicals has the potential to positively impact the South African economy, but is reliant on the development of low-cost conversion technology. Perhaps the most important progress to be made is the development of “consolidated bioprocessing” (CBP). CBP refers to the conversion of pretreated biomass into desired product(s) in a single process step with either a single organism or consortium of organisms and without the addition of cellulase enzymes. Among the microbial hosts considered for CBP development, Saccharomyces cerevisiae has received significant interest from the biotechnology community as the yeast preferred for ethanol production. The major advantages of S. cerevisiae include high ethanol productivity and tolerance, as well as a well-developed gene expression system. Since S. cerevisiae is non-cellulolytic, the functional expression of at least three groups of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21) is a prerequisite for cellulose conversion via CBP. The endo- and exoglucanases act synergistically to efficiently degrade cellulose to soluble cellodextrins and cellobiose, whereas the β-glucosidases catalyze the conversion of the soluble cellulose hydrolysis products to glucose. This study focuses on the efficient utilization of cellobiose by recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Since it is generally accepted that S. cerevisiae do not produce a dedicated cellobiose permease/transporter, the obvious strategy was to produce a secretable β-glucosidase that will catalyze the hydrolysis of cellobiose to glucose extracellularly. β-Glucosidase genes of various fungal origins were isolated and heterologously expressed in S. cerevisiae. The mature peptide sequence of the respective β-glucosidases were fused to the secretion signal of the Trichoderma reesei xyn2 gene and expressed constitutively from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The maximum specific growth rates (μmax) of the recombinant strains were compared during batch cultivation in high-performance bioreactors. S. cerevisiae secreting the recombinant Saccharomycopsis fibuligera BGL1 enzyme was identified as the best strain and grew at 0.23 h-1 on cellobiose (compared to 0.29 h-1 on glucose). More significantly, was the ability of this strain to anaerobically ferment cellobiose at 0.18 h-1 (compared to 0.25 h-1 on glucose). However, extracellular cellobiose hydrolysis has two major disadvantages, namely glucose’s inhibitory effect on the activity of cellulase enzymes as well as the increased risk of contamination associated with external glucose release. In an alternative approach, the secretion signal from the S. fibuligera β-glucosidase (BGL1) was removed and expressed constitutively from the above-mentioned multi-copy yeast expression vector. Consequently, the BGL1 enzyme was functionally produced within the intracellular space of the recombinant S. cerevisiae strain. A strategy employing continuous selection pressure was used to adapt the native S. cerevisiae disaccharide transport system(s) for cellobiose uptake and subsequent intracellular utilization. RNA Bio-Dot results revealed the induction of the native α-glucoside (AGT1) and maltose (MAL) transporters in the adapted strain, capable of transporting and utilizing cellobiose intracellularly. Aerobic batch cultivation of the strain resulted in a μmax of 0.17 h-1 and 0.30 h-1 when grown in cellobiose- and cellobiose/maltose-medium, respectively. The addition of maltose significantly improved the uptake of cellobiose, suggesting that cellobiose transport (via the combined action of the maltose permease and α-glucosidase transporter) is the rate-limiting step when the adapted strain is grown on cellobiose as sole carbon source. In agreement with the increased μmax value, the substrate consumption rate also improved significantly from 0.25 g.g DW-1.h-1 when grown on cellobiose to 0.37 g.g DW-1.h-1 upon addition of maltose to the medium. The adapted strain also displayed several interesting phenotypical characteristics, for example, flocculation, pseudohyphal growth and biofilm-formation. These features resemble some of the properties associated with the highly efficient cellulase enzyme systems of cellulosome-producing anaerobes. Recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Both recombinant strains are of particular interest when the final goal of industrial-scale ethanol production from cellulosic waste is considered. However, the latter strain’s ability to efficiently remove cellobiose from the extracellular space together with its flocculating, pseudohyphae- and biofilm-forming properties can be an additional advantage when the recombinant S. cerevisiae strain is considered as a potential host for future CBP technology. / AFRIKAANSE OPSOMMING: Die omskakeling van sellulose-bevattende biomassa na brandstof en chemikalieë beskik oor die potensiaal om die Suid-Afrikaanse ekonomie positief te beïnvloed, indien bekostigbare tegnologie ontwikkel word. Die merkwaardigste vordering tot dusvêr kon in die ontwikkeling van “gekonsolideerde bioprosessering” (CBP) wees. CBP verwys na die eenstap-omskakeling van voorafbehandelde biomassa na gewenste produkte met behulp van ‘n enkele organisme of ‘n konsortium van organismes sonder die byvoeging van sellulase ensieme. Onder die mikrobiese gashere wat oorweeg word vir CBP-ontwikkeling, het Saccharomyces cerevisiae as die voorkeur gis vir etanolproduksie troot belangstelling by die biotegnologie-gemeenskap ontlok. Die voordele van S. cerevisiae sluit in hoë etanol-produktiwiteit en toleransie, tesame met ‘n goed ontwikkelde geen-uitdrukkingsisteem. Aangesien S. cerevisiae nie sellulose kan benut nie, is die funksionele uitdrukking van ten minste drie groepe ensieme, naamlik endoglukanases (EC 3.2.1.4); eksoglukanases (EC 3.2.1.91) en β-glukosidases (EC 3.2.1.21), ‘n voorvereiste vir die omskakeling van sellulose via CBP. Die sinergistiese werking van endo- en eksoglukanases word benodig vir die effektiewe afbraak van sellulose tot oplosbare sello-oligosakkariede en sellobiose, waarna β-glukosidases die finale omskakeling van die oplosbare sellulose-afbraak produkte na glukose kataliseer. Hierdie studie fokus op die effektiewe benutting van sellobiose m.b.v. rekombinante S. cerevisiae-rasse met die vermoeë om sellobiose ekstrasellulêr af te breek of dit op te neem en intrasellulêr te benut. Aangesien dit algemeen aanvaar word dat S. cerevisiae nie ‘n toegewyde sellobiosepermease/ transporter produseer nie, was die mees voor-die-hand-liggende strategie die produksie van ‘n β-glukosidase wat uitgeskei word om sodoende die ekstrasellulêre hidroliese van sellobiose na glukose te kataliseer. β-Glukosidase gene is vanaf verskeie fungi geïsoleer en daaropvolgend in S. cerevisiae uitgedruk. Die geprosesseerde peptiedvolgorde van die onderskeie β-glukosidases is met die sekresiesein van die Trichoderma reesei xyn2-geen verenig en konstitutief vanaf ‘n multikopie-gisuitdrukkingsvektor onder transkripsionele beheer van die S. cerevisiae PGK1 promotor en termineerder uitgedruk. Die gevolglike rekombinante ensieme is op grond van hul pH en temperatuur optima, asook kinetiese eienskappe, gekarakteriseer. Die maksimum spesifieke groeitempos (μmax) van die rekombinante rasse is gedurende aankweking in hoë-verrigting bioreaktors vergelyk. Die S. cerevisiae ras wat die rekombinante Saccharomycopsis fibuligera BGL1 ensiem uitskei, was as the beste ras geïdentifiseer en kon teen 0.23 h-1 op sellobiose (vergeleke met 0.29 h-1 op glukose) groei. Meer noemenswaardig is the ras se vermoë om sellobiose anaërobies teen 0.18 h-1 (vergeleke met 0.25 h-1 op glukose) te fermenteer. Ekstrasellulêre sellobiose-hidroliese het twee groot nadele, naamlik glukose se onderdrukkende effek op die aktiwiteit van sellulase ensieme, asook die verhoogde risiko van kontaminasie wat gepaard gaan met die glukose wat ekstern vrygestel word. ’n Alternatiewe benadering waarin die sekresiesein van die S. fibuligera β-glucosidase (BGL1) verwyder en konstitutief uitgedruk is vanaf die bogenoemde multi-kopie gisuitrukkingsvektor, is gevolg. Die funksionele BGL1 ensiem is gevolglik binne-in die intrasellulêre ruimte van die rekombinante S. cerevisiae ras geproduseer. Kontinûe selektiewe druk is gebruik om die oorspronklike S. cerevisiae disakkaried-transportsisteme vir sellobiose-opname and daaropvolgende intrasellulêre benutting aan te pas. RNA Bio-Dot resultate het gewys dat die oorspronklike α-glukosied (AGT1) en maltose (MAL) transporters in die aangepaste ras, wat in staat is om sellobiose op te neem en intrasellulêr te benut, geïnduseer is. Aërobiese kweking van die geselekteerde ras het gedui dat die ras teen 0.17 h-1 en 0.30 h-1 groei in onderskeidelik sellobiose en sellobiose/maltose-medium. Die byvoeging van maltose het die opname van sellobiose betekenisvol verbeter, waarna aangeneem is dat sellobiose transport (via die gekombineerde werking van die maltose permease en α-glukosidase transporter) die beperkende stap gedurende groei van die geselekteerde ras op sellobiose as enigste koolstofbron is. In ooreenstemming hiermee, het die substraatbenuttingstempo ook betekenisvol toegeneem van 0.25 g.g DW-1.h-1, gedurende groei op sellobiose, tot 0.37 g.g DW-1.h-1 wanneer maltose by die medium gevoeg word. Die geselekteerde ras het ook verskeie interessante fenotipiese kenmerke getoon, byvoorbeeld flokkulasie, pseudohife- en biofilm-vorming. Hierdie eienskappe kom ooreen met sommige van die kenmerke wat met die hoogs effektiewe sellulase ensiem-sisteme van sellulosomeproduserende anaerobe geassosieer word. Hierdie studie beskryf die suksesvolle konstruksie van ‘n rekombinante S. cerevisiae ras met die vermoë om sellobiose ekstrasellulêr af te breek of om dit op te neem en intrasellulêr te benut. Beide rekombinante rasse is van wesenlike belang indien die einddoel van industriële-skaal etanolproduksie vanaf selluloseafval oorweeg word. Die laasgenoemde ras se vermoë om sellobiose effektief uit die ekstrasellulêre ruimte te verwyder tesame met die flokkulasie, pseudohife- en biofilm-vormings eienskappe kan ‘n addisionele voordeel inhou, indien die rekombinante S. cerevisiae ras as ‘n potensiële gasheer vir toekomstige CBP-tegnologie oorweeg word.

Cellulose from energy crops or agriculture residues can be utilized as a sustainable energy resource to produce biofuels such as ethanol. The process of converting cellulose into solvents and biofuels requires the saccharification of cellulose into soluble, fermentable sugars. However, challenges to cellulosic biofuel production include increasing the activity of cellulose-degrading enzymes (cellulases) and increasing solvent (ethanol) yield while minimizing the co-production of organic acids. This work applies novel surface analysis techniques and fermentation reactor perturbations to quantify, manipulate, and model enzymatic and metabolic processes critical to the efficient production of cellulosic biofuels. Surface analysis techniques utilizing cellulose thin film as the model substrate are developed to quantify the kinetics of cellulose degradation by cellulase as well as the interactions with cellulase at the interfacial level. Quartz Crystal Microbalance with Dissipation (QCM-D) is utilized to monitor the change in mass of model cellulose thin films cast. The time-dependent frequency response of the QCM simultaneously measures both enzyme adsorption and hydrolysis of the cellulose thin film by fungal cellulases, in which a significant reduction in the extent of hydrolysis can be observed with increasing cellobiose concentrations. A mechanistic enzyme reaction scheme is successfully applied to the QCM frequency response for the first time, describing adsorption/desorption and hydrolysis events of the enzyme, inhibitor, and enzyme/inhibitor complexes. The effect of fungal cellulase concentration on hydrolysis is tested using the QCM frequency response of cellulose thin films. Atomic Force Microscopy (AFM) is also applied for the first time to the whole cell cellulases of the bacterium C. thermocellum, where the effect of temperature on hydrolysis activity is quantified. Fermentation of soluble sugars to desirable products requires the optimization of product yield and selectivity of the cellulolytic bacterium, Clostridium thermocellum. Metabolic tools to map the phenotype toward desirable solvent production are developed through environmental perturbation. A significant change in product selectivity toward ethanol production is achieved with exogenous hydrogen and the addition of hydrogenase inhibitors (e.g. methyl viologen). These results demonstrate compensatory product formation in which the shift in metabolic activity can be achieved through environmental perturbation without permanent change in the organism’s genome.

This project aimed investigated cellulase in-situ production for large-scale on-farm production of lignocellulosic biofuel. Cellulase activity and glucose released by T. reesei with corn stover and wheat bran as co-substrates for solid state cultivation (SSC) were examined. Co-cultivation has previously increased T. reesei cellulase, but corn stover and wheat bran have not been co-cultivated (Dhillon, Oberoi et al. 2011). This work compared cellulase activity and glucose concentration of corn stover co-cultivated with 0-40% wheat bran in high solids. Samples with at least 20% wheat bran exhibited increased cellulase activity. However, the average glucose concentration without wheat bran was 3.29 g/L compared to 16.7 g/L with wheat bran. Glucose released by T. reesei on pretreated corn stover with 0-40% wheat bran was compared at the optimal temperatures for fungal growth and for cellulase activity after SSC. Previous research has rarely used cellulase from SSC to hydrolyze lignocellulose. Following SSC of T. reesei at 30°C for seven days, samples were warmed to 50°C for five days. Glucose concentration increased to 12.1 and 32.7 g/L for samples with and without wheat bran. This strategy could reduce lignocellulosic fuel production costs by eliminating need for commercial cellulase and is promising for efficient cellulose hydrolysis.

A Floresta Amazônica, o maior bioma brasileiro, é caracterizado pela ampla diversidade e heterogeneidade de seus ecossistemas. Os solos amazônicos, em geral, abrigam elevada diversidade microbiana que desempenha papeis importantes na ciclagem de nutrientes, mediante a decomposição da matéria orgânica. Dentre os micro-organismos, os fungos se destacam como os principais agentes envolvidos na biodegradação. Esses micro-organismos produzem um coquetel de enzimas do complexo celulolítico que são de grande importância biotecnológica. Desse modo, diante da importância econômica dos fungos, o presente trabalho propôs, acessar fungo do gênero Trichoderma, obtidos de solos da Floresta Amazônica, na busca por linhagens com alto potencial celulolítico. Assim, a partir de amostras de solos coletados em doze pontos da floresta foram obtidos 151 isolados de Trichoderma spp. O sequenciamento do gene que codifica para o de elongação da transcrição alfa-1, evidenciou a prevalência de sete espécies de Trichoderma. Do total de isolados, as mais abundantes foram: Trichoderma spirale (37%), Trichoderma strigosum (22%), Trichoderma harzianum (18%) e Trichoderma asperellum (17%). As linhagens foram submetidas a triagem para atividade celulolítica. Duas linhagens, T. harzianum AMS 23.14 e T. harzianum AMS 29.14 apresentaram atividade enzimática superior ao padrão, T. reesei RUT C-30 e foram submetidas a avaliação da atividade enzimática em diferentes condições de cultivos (bagaço de cana-deaçúcar tratado e não tratado, em dois pHs: 3 e 5). Foi observado atividade superior das linhagens amazônicas para as três enzimas estudadas (endoglucanase, exoglucanase e β-glicosidase), quando comparadas à linhagem padrão utilizada. A atividade enzimática foi positivamente influenciada pelo pH ácido, assim como pelo substrato não tratado. A partir destes resultados é notório que as linhagens isoladas desse bioma possuem grande potencial de atividade celulolítica, e que estudos mais aprofundados podem proporcionar o futuro emprego desses fungos em diversas áreas industriais. / The Amazon Rain Forest, the largest Brazilian biome, is characterized by the wide diversity and heterogeneity of its ecosystems. Amazonian soils, in general, harbor high microbial diversity that plays important roles in the cycling of nutrients, through the decomposition of organic matter. Among the microorganisms, fungi stand out as biodegradation agents. These microorganisms produce cellulolytic enzymes that are of great economic and biotechnological importance. The present work proposed to access the fungal population of the genus Trichoderma, isolated from Amazonian Forest soils, in the search for lineages with high potential of cellulolytic activity. Twelve soil samples were collected, resulting in the isolation of 151 strains of Trichoderma spp. Gene sequencing of the alpha-1 transcription elongation factor region with the EF1 and TEFR primers evidenced the prevalence of seven Trichoderma species. Of the total isolates, Trichoderma spirale (37%), Trichoderma strigosum (22%), Trichoderma harzianum (18%) and Trichoderma asperellum (17%) were the most abundant. The strains were screened for qualitative cellulolytic activity in solid medium and quantitative in liquid medium. Two strains, T. harzianum AMS 23.14 and T. harzianum AMS 29.14 showed enzymatic activity superior to T. reesei RUT C-30, and were sent to the evaluation of the enzymatic activity in different conditions (sugarcane bagasse treated and untreated, in two pHs: 3 and 5). Superior activity of the Amazonian strains was observed for the three enzymes studied (endoglucanase, exoglucanase and β-glucosidase) when compared to the T. reesei RUT C-30 standard. The enzymatic activity was positively influenced by pH 3, as well as by the untreated substrate. From these results, it is well known that isolated strains of the Amazonian environment have a great potential for cellulolytic activity, and that further studies may provide the future employment of these strains in several areas of industry.

A Floresta Amazônica, o maior bioma brasileiro, é caracterizado pela ampla diversidade e heterogeneidade de seus ecossistemas. Os solos amazônicos, em geral, abrigam elevada diversidade microbiana que desempenha papeis importantes na ciclagem de nutrientes, mediante a decomposição da matéria orgânica. Dentre os micro-organismos, os fungos se destacam como os principais agentes envolvidos na biodegradação. Esses micro-organismos produzem um coquetel de enzimas do complexo celulolítico que são de grande importância biotecnológica. Desse modo, diante da importância econômica dos fungos, o presente trabalho propôs, acessar fungo do gênero Trichoderma, obtidos de solos da Floresta Amazônica, na busca por linhagens com alto potencial celulolítico. Assim, a partir de amostras de solos coletados em doze pontos da floresta foram obtidos 151 isolados de Trichoderma spp. O sequenciamento do gene que codifica para o de elongação da transcrição alfa-1, evidenciou a prevalência de sete espécies de Trichoderma. Do total de isolados, as mais abundantes foram: Trichoderma spirale (37%), Trichoderma strigosum (22%), Trichoderma harzianum (18%) e Trichoderma asperellum (17%). As linhagens foram submetidas a triagem para atividade celulolítica. Duas linhagens, T. harzianum AMS 23.14 e T. harzianum AMS 29.14 apresentaram atividade enzimática superior ao padrão, T. reesei RUT C-30 e foram submetidas a avaliação da atividade enzimática em diferentes condições de cultivos (bagaço de cana-deaçúcar tratado e não tratado, em dois pHs: 3 e 5). Foi observado atividade superior das linhagens amazônicas para as três enzimas estudadas (endoglucanase, exoglucanase e β-glicosidase), quando comparadas à linhagem padrão utilizada. A atividade enzimática foi positivamente influenciada pelo pH ácido, assim como pelo substrato não tratado. A partir destes resultados é notório que as linhagens isoladas desse bioma possuem grande potencial de atividade celulolítica, e que estudos mais aprofundados podem proporcionar o futuro emprego desses fungos em diversas áreas industriais. / The Amazon Rain Forest, the largest Brazilian biome, is characterized by the wide diversity and heterogeneity of its ecosystems. Amazonian soils, in general, harbor high microbial diversity that plays important roles in the cycling of nutrients, through the decomposition of organic matter. Among the microorganisms, fungi stand out as biodegradation agents. These microorganisms produce cellulolytic enzymes that are of great economic and biotechnological importance. The present work proposed to access the fungal population of the genus Trichoderma, isolated from Amazonian Forest soils, in the search for lineages with high potential of cellulolytic activity. Twelve soil samples were collected, resulting in the isolation of 151 strains of Trichoderma spp. Gene sequencing of the alpha-1 transcription elongation factor region with the EF1 and TEFR primers evidenced the prevalence of seven Trichoderma species. Of the total isolates, Trichoderma spirale (37%), Trichoderma strigosum (22%), Trichoderma harzianum (18%) and Trichoderma asperellum (17%) were the most abundant. The strains were screened for qualitative cellulolytic activity in solid medium and quantitative in liquid medium. Two strains, T. harzianum AMS 23.14 and T. harzianum AMS 29.14 showed enzymatic activity superior to T. reesei RUT C-30, and were sent to the evaluation of the enzymatic activity in different conditions (sugarcane bagasse treated and untreated, in two pHs: 3 and 5). Superior activity of the Amazonian strains was observed for the three enzymes studied (endoglucanase, exoglucanase and β-glucosidase) when compared to the T. reesei RUT C-30 standard. The enzymatic activity was positively influenced by pH 3, as well as by the untreated substrate. From these results, it is well known that isolated strains of the Amazonian environment have a great potential for cellulolytic activity, and that further studies may provide the future employment of these strains in several areas of industry.

A importância do estudo das celulases não se limita à aquisição de conhecimento científico, mas também ao grande potencial biotecnológico que elas representam. Isso se deve ao fato da celulose ser a molécula mais abundante presente na natureza e prover uma vasta gama de produtos e processos sustentáveis. Muitas famílias de celulases já foram bem caracterizadas, enquanto outras permanecem ainda desconhecidas. Dentre estas últimas, a família 45 das hidrolases de glicosídeos é a família de celulases fúngicas menos caracterizada tanto estruturalmente quanto funcionalmente. Recentemente foi proposta a divisão dessa família em três subfamílias e, até agora, apenas membros da subfamília A tiveram enzimas estruturalmente elucidadas. Nesse trabalho reportamos a estrutura cristalográfica da proteína recombinante endoglucanase de Phanerochaete chrysosporium (PcCel45A), a primeira das hidrolases de glicosídeos da subfamília C, e seu complexo com celobiose a 1,4 Å e 1,7 Å de resolução, respectivamente. A PcCel45A é uma enzima de domínio único, com uma estrutura em β-barril e seu empacotamento geral remete ao formato de âncora. O sítio ativo da enzima forma um longo sulco na superfície da estrutura, sendo que o seu centro catalítico é diferente das outras enzimas publicadas dessa família e o aspartato catalítico, que atua como aceptor de próton na reação de inversão, (Asp10) não é conservado. Adicionalmente, a estrutura cristalográfica dessa enzima apresenta mais similaridades com as β-expansinas (proteínas de plantas) e transglicosilases líticas (proteínas que clivam o peptidoglicano de bactérias) do que com as outras representantes da família 45, o que a torna ainda mais singular. Para entendermos melhor seu funcionamento foram realizadas mutações sítio-dirigidas nos principais resíduos do sítio ativo. O Asp121, conhecido por participar da reação de inversão das outras enzimas da família como doador de próton, mostrou-se essencial para a atividade da enzima, enquanto que outros resíduos conservados como a Tyr25, o Trp161 e o Asp92 afetaram, mas não aniquilaram a atividade da enzima, apresentando aproximadamente 20%, 50% e 10% da atividade da enzima nativa, respectivamente. / The importance of the study of the cellulases is not limited to generating significant scientific knowledge, since these enzymes represents an enormous potential in biotechnology. This is partly because cellulose is the most abundant molecule in nature and provides a wide range of products and sustainable process. Many cellulases families have been well characterized, while others still remain unknown. Among them, the glycoside hydrolase family 45 is the least well characterized both structurally and functionally, between fungal cellulases. It was recently proposed the subdivision of this family into three subfamilies, with structural information available only for subfamily A. In this work, we report the chrystallographic structure of the recombinant endoglucanase from Phanerochaete chrysosporium (PcCel45A), the first GH45 subfamily C and its complex with cellobiose at 1.4 Å and 1.7 Å respectively. The PcCel45A is a single domain enzyme, which has a β-barrel structure with the overall shape resembling an anchor. The active site of the enzyme has a long cleft on the surface, being remarkably different from those members of subfamily A, and the catalytic aspartate responsible for acting as proton acceptor (Asp10) is not present. Additionally, the chrystallographic structure of this enzyme has shown more similarity with β -expansins (plant proteins) and lytic transglycosylase (proteins that cleave the peptidoglycan of bacteria) than others representants of family 45, which makes it more singular. For a better understanding of its function, we perform pontual mutations in the main residues from active site. The Asp121, known for acting as proton acceptor in the inversion reaction of others enzymes, proved to be essential for the enzyme activity, while others conserved residues as Tyr25, Trp161 and Asp92 affected but not annihilated the enzyme activity, leaving approximately 20%, 50% and 10% of the native enzyme activity.