ARTIGOS PUBLICADOS COMO PRIMEIRO AUTOR OU

160

Artigo 1

Deciphering the synergism of endogenous glycoside hydrolase families 1 and 9 from Coptotermes gestroi

João Paulo L. Franco Cairoa,b_{, Leandro C. Oliveira}a,e_{, Cristiane A. Uchima}a_,

Thabata M. Alvareza_{, Ana Paula da S. Citadini}a_{, Júnio Cota}a_{, Flávia Costa Leonardo}b,c_, Ana M. Costa-Leonardod_{, Marcelo F. Carazzolle}b_{, Fernando F. Costa}c_,

Gonçalo A.G. Pereirab_{, Fabio M. Squina}a,*

a_{Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Rua Giuseppe Máximo} Scolfaro, no 10000, 13083-970 Campinas, SP, Brazil

b_{Laboratório de Genômica e Expressão (LGE), Departamento de Genética, Evolução e Bioagentes da Universidade Estadual de Campinas (UNICAMP),} Campinas, Brazil

c_{Centro de Hematologia e Hemoterapia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil} d_{Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Rio Claro, Brazil}

e_{Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas (IBILCE), Universidade Estadual Paulista (UNESP), São José do Rio Preto, SP, Brazil}

a r t i c l e i n f o

Article history: Received 24 April 2013 Received in revised form 24 July 2013 Accepted 25 July 2013 Keywords: Termite Glycoside hydrolase Molecular docking Coptotermes gestroi Synergistic interaction a b s t r a c t

Termites can degrade up to 90% of the lignocellulose they ingest using a repertoire of endogenous and symbiotic degrading enzymes. Termites have been shown to secrete two main glycoside hydrolases, which are GH1 (EC 3.2.1.21) and GH9 (EC 3.2.1.4) members. However, the molecular mechanism for lignocellulose degradation by these enzymes remains poorly understood. The present study was con- ducted to understand the synergistic relationship between GH9 (CgEG1) and GH1 (CgBG1) from Cop- totermes gestroi,which is considered the major urban pest of São Paulo State in Brazil. The goal of this work was to decipher the mode of operation of CgEG1 and CgBG1 through a comprehensive biochemical analysis and molecular docking studies. There was outstanding degree of synergy in degrading glucose polymers for the production of glucose as a result of the endo-b-1,4-glucosidase and exo-b-1,4- glucosidase degradation capability of CgEG1 in concert with the high catalytic performance of CgBG1, which rapidly converts the oligomers into glucose. Our data not only provide an increased compre- hension regarding the synergistic mechanism of these two enzymes for cellulose sacchariﬁcation but also give insight about the role of these two enzymes in termite biology, which can provide the foun- dation for the development of a number of important applied research topics, such as the control of termites as pests as well as the development of technologies for lignocellulose-to-bioproduct applications.

1. Introduction

Termites are considered the smallest bioreactor in the world (Brune, 1998). The termite’s digestome can degrade up to 90% of the

lignocellulosic biomass ingested (Katsumata et al., 2007) using a repertoire of endogenous and symbiotic degrading enzymes, accessory proteins, and putative ligninases (Chandrasekharaiah et al., 2011; Coy et al., 2010; Hongoh, 2011; Tartar et al., 2009; Todaka et al., 2010, 2007; Warnecke et al., 2007). The termite gut is generally divided in three compartments: foregut, midgut and hindgut. After the termites ingest lignocellulose material, the biomass is reduced to about 20mm by their mandibles (Fujita et al., 2010), then further exposing the material to the degradation of endogenous enzymes, such as endo-b-1,4-glucosidase (GH9),b- glucosidase (GH1) (Brune and Ohkuma, 2011; Ohkuma, 2003; Ohkuma et al., 2008), laccases and esterases in the foregut (Coy et al., 2010; Sethi et al., 2013; Wheeler et al., 2010). These Abbreviations:APTS, 8-aminopyreno-1,3,6-trisulfonic acid; BIN, integral sugar-

cane bagasse; CD, circular dichroism; CMC, carboxymethyl cellulose; CZE, capillary zone electrophoresis; DNS, 3,5-dinitrosalicylic acid; DS, degree of synergism; GH, glycoside hydrolase; GOD, glucose oxidase; IMAC, immobilized metal afﬁnity chromatography; PASB, phosphoric acid pretreated sugarcane bagasse; p-NP, p- nitrophenyl.

*Corresponding author. Tel.: þ55 19 35183111; fax: þ55 19 35183164. E-mail addresses:fabio.squina@bioetanol.org.br,fmsquina@gmail.com(F.M. Squina).

Contents lists available atScienceDirect

Insect Biochemistry and Molecular Biology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i b m b

http://dx.doi.org/10.1016/j.ibmb.2013.07.007

Artigo 2

Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse

by supplementation with pectinase anda-L-arabinofuranosidase

Priscila da Silva Delabonaa,_⇑_{, Júnio Cota}a_{, Zaira Bruna Hoffmam}a_{, Douglas Antonio Alvaredo Paixão}a_, Cristiane Sanchez Farinasb_{, João Paulo Lourenço Franco Cairo}a_{, Deise Juliana Lima}a_{, Fábio Marcio Squina}a_, Roberto Rullera_{, José Geraldo da Cruz Pradella}a

a_{Brazilian Bioethanol Science and Technology Laboratory – CTBE, Rua Giuseppe Maximo Scolfaro 10000, Pólo II de Alta Tecnologia, Caixa Postal 6192, CEP 13083-970,}

Campinas, São Paulo, Brazil

b_{Embrapa Instrumentation, Rua XV de Novembro 1452, CEP 13560-970, São Carlos, São Paulo, Brazil}

h i g h l i g h t s

"The Amazon rainforest Trichoderma harzianum is a potential candidate for saccharification of plant biomass. "Important roles for pectinase anda-L-arabinofuranosidase in the enzymatic synergies.

"Sugarcane bagasse hydrolysis is more effective and competitive for second generation ethanol.

a r t i c l e i n f o

Article history: Received 30 July 2012

Received in revised form 27 November 2012 Accepted 14 December 2012

Available online 22 December 2012

Keywords: Accessory enzymes Trichoderma harzianum Pectinase a-L-arabinofuranosidase Enzymatic hydrolysis a b s t r a c t

Supplementation of cellulase cocktails with accessory enzymes can contribute to a higher hydrolytic capacity in releasing fermentable sugars from plant biomass. This study investigated which enzymes were complementary to the enzyme set of Trichoderma harzianum in the degradation of sugarcane bagasse. Specific activities of T. harzianum extract on different substrates were compared with the extracts of Penicillium echinulatum and Trichoderma reesei, and two commercial cellulase preparations. Complementary analysis of the secretome of T. harzianum was also used to identify which enzymes were produced during growth on pretreated sugarcane bagasse. These analyses enabled the selection of the enzymes pectinase anda-L-arabinofuranosidase (AF) to be further investigated as supplements to the T. harzianumextract. The effect of enzyme supplementation on the efficiency of sugarcane bagasse saccharification was evaluated using response surface methodology. The supplementation of T. harzianum enzymatic extract with pectinase and AF increased the efficiency of hydrolysis by up to 116%.

1. Introduction

The conversion of lignocellulosic biomass into fuels and other chemicals can be achieved using a multi-enzyme system acting in synergy. An enzymatic cocktail containing different enzymes involved in the hydrolysis of each part of the lignocellulosic structure is crucial to increase enzymatic hydrolysis (EH) yields (Alvira et al., 2011; Berlin et al., 2007; Gao et al., 2011). In addition, the viable bioconversion of biomass requires not only that the cost of the enzymes be reduced, but also that improvements be made in the effectiveness of the enzymatic extracts used in the hydrolysis mix- ture. These aspects are highly dependent on both the raw material

and the pretreatment employed (Delabona et al., 2012a; Jorgensen and Olsson, 2006; Sorensen et al., 2011; Sukumaran et al., 2009). On-site production of enzymes can help to reduce enzyme costs since there is less need to stabilize the enzyme preparations, avoid- ing costs associated with transport and storage for long periods. Furthermore, the use of the same lignocellulosic biomass for enzyme production and hydrolysis could reduce the production costs of bioethanol, since both process could be co-located and share infrastructure and utilities (Delabona et al., 2012a). The on-site enzyme production using a split stream from the bioethanol process as part of the fermentation medium could be an attractive alternative (Kovacs et al., 2009; Sorensen et al., 2011). Moreover, the production of enzymes using as carbon source the same lignocellulosic material that will be used in the hydrolysis has shown that these enzyme preparations can present better performance (Delabona et al., 2012a; Jorgensen and Olsson, 2006).

http://dx.doi.org/10.1016/j.biortech.2012.12.105

⇑Corresponding author. Tel.: +55 (19) 3518 3154; fax: +55 (19) 3518 3104. E-mail address:priscila.delabona@bioetanol.org.br(P.S. Delabona).

Bioresource Technology 131 (2013) 500–507

Contents lists available atSciVerse ScienceDirect

Bioresource Technology

162

Artigo 3

O R I G I N A L A R T I C L E

The characterization of a thermostable and cambialistic superoxide dismutase from Thermus filiformis

F. Mandelli1,2_{, J.P.L. Franco Cairo}1_{, A.P.S. Citadini}1_{, F. B€}_uchli1_{, T.M. Alvarez}1_{, R.J. Oliveira}3_,

V.B.P. Leite3_{, A.F. Paes Leme}4_{, A.Z. Mercadante}2_{and F.M. Squina}1

1 Laborat"orio Nacional de Ci^encia e Tecnologia do Bioetanol (CTBE), do Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil

2 Departamento de Ci^encia de Alimentos da Faculdade de Engenharia de Alimentos, UNICAMP, Campinas, Brazil

3 Departamento de F"ısica, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista, S~ao Jos"e do Rio Preto, Brazil 4 Laborat"orio Nacional de Biociências (LNBio), do Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil

Significance and Impact of the Study: This manuscript describes the expression and characterization of a superoxide dismutase (SOD) from Thermus filiformis with thermophilic and cambialistic characteristics. The SODs are among the most potent antioxidants known in nature, and their stability and pharmacoki- netics can vary widely in accordance to their biological source. Although the currently clinical research work has been focused on human and bovine SODs, alternative sources may become more biotechnological attractive in the near future. Our study brings new insights for the research field of antioxidant enzymes with potential application on pharmaceutical, cosmetics and food formulations.

Keywords

3-D model, cambialistic enzyme, circular dichroism, nitroblue tetrazolium, pyrogallol autoxidation.

Correspondence

F.M. Squina, Laborat"orio Nacional de Ci^encia e Tecnologia do Bioetanol (CTBE), do Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Rua Giuseppe M"aximo Scolfaro, nº 10000, Campinas, SP-13083-970, Brazil. E-mail: fabio.squina@bioetanol.org.br 2012/2317: received 27 December 2012, revised 15 March 2013 and accepted 15 March 2013

doi:10.1111/lam.12071

Abstract

The superoxide dismutase (TfSOD) gene from the extremely thermophilic bacterium Thermus filiformis was cloned and expressed at high levels in mesophilic host. The purified enzyme displayed approximately 25 kDa band in the SDS-PAGE, which was further confirmed as TfSOD by mass spectrometry. The TfSOD was characterized as a cambialistic enzyme once it had enzymatic activity with either manganese or iron as cofactor. TfSOD showed thermostability at 65, 70 and 80°C. The amount of enzyme required to inhibit 50% of pyrogallol autoxidation was 0!41, 0!56 and 13!73 mg at 65, 70 and 80°C, respectively. According to the circular dichroism (CD) spectra data, the secondary structure was progressively lost after increasing the temperature above 70°C. The 3-dimensional model of TfSOD with the predicted cofactor binding corroborated with functional and CD analysis.

Introduction

Antioxidant enzymes such as superoxide dismutase (SOD) and catalase protect living cells from reactive oxygen and nitrogen species that are responsible for oxidative damage of essential components of cell structure (Pantazaki et al. 2002). Superoxide dismutases are a class of antioxidant defence metalloenzymes that disproportionate superoxide radical ion into molecular oxygen and hydrogen peroxide (Whittaker and Whittaker 1999). Due to its antioxidative effects, SOD has been widely applied in medical treatments,

as well as cosmetic, food, agricultural and chemical industries (Liu et al. 2011). SODs have recently found applications as supplementation to prevent or reverse the adverse effect of cardiovascular diseases, ageing, infertility, neuro- logical disorders, ischaemia–reperfusion injury, transplant rejection, autoimmune diseases, rheumatoid arthritis, diabetes, asthma, septic shock-induced tissue injury and cancer, as well as in the pharmaceutical and cosmetic industries (Bafana et al. 2011).

Once thermal denaturation is a common cause of enzyme inactivation in the industry, one of the major

Artigo 4

Structure and Function of a Novel Cellulase 5 from Sugarcane Soil Metagenome

Thabata M. Alvarez1☯_{, Joice H. Paiva}2☯_{, Diego M. Ruiz}2_{, João Paulo L. F. Cairo}1_{, Isabela O. Pereira}1_,

Douglas A. A. Paixão1_{, Rodrigo F. de Almeida}1_{, Celisa C. C. Tonoli}2_{, Roberto Ruller}1_{, Camila R. Santos}2_,

Fabio M. Squina1*_{, Mario T. Murakami}2*

1 Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, São Paulo, Brazil, 2 Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, São Paulo, Brazil

Abstract

Cellulases play a key role in enzymatic routes for degradation of plant cell-wall polysaccharides into simple and economically-relevant sugars. However, their low performance on complex substrates and reduced stability under industrial conditions remain the main obstacle for the large-scale production of cellulose-derived products and biofuels. Thus, in this study a novel cellulase with unusual catalytic properties from sugarcane soil metagenome (CelE1) was isolated and characterized. The polypeptide deduced from the celE1 gene encodes a unique glycoside hydrolase domain belonging to GH5 family. The recombinant enzyme was active on both carboxymethyl cellulose and β-glucan with an endo-acting mode according to capillary electrophoretic analysis of cleavage products. CelE1 showed optimum hydrolytic activity at pH 7.0 and 50 °C with remarkable activity at alkaline conditions that is attractive for industrial applications in which conventional acidic cellulases are not suitable. Moreover, its three- dimensional structure was determined at 1.8 Å resolution that allowed the identification of an insertion of eight residues in the β8-α8 loop of the catalytic domain of CelE1, which is not conserved in its psychrophilic orthologs. This 8-residue-long segment is a prominent and distinguishing feature of thermotolerant cellulases 5 suggesting that it might be involved with thermal stability. Based on its unconventional characteristics, CelE1 could be potentially employed in biotechnological processes that require thermotolerant and alkaline cellulases.

Citation: Alvarez TM, Paiva JH, Ruiz DM, Cairo JPLF, Pereira IO, et al. (2013) Structure and Function of a Novel Cellulase 5 from Sugarcane Soil Metagenome. PLoS ONE 8(12): e83635. doi:10.1371/journal.pone.0083635

Editor: Vickery Arcus, University of Waikato, New Zealand

Received August 26, 2013; Accepted November 4, 2013; Published December 17, 2013

Copyright: © 2013 Alvarez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the Brazilian Research Agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grants number: 09/08312-6 and 13/13309-0 to MTM, 2008/58037-9 to FMS, 2010/11469-1 to TMA, 2012/18559-5 to RG, 2011/20977-3 to JPLFC; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grants number 478059/2009-4 and 486841/2012-0 awarded to MTM; and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. * E-mail: mario.murakami@lnbio.cnpem.br (MTM); fabio.squina@bioethanol.org.br (FMS) ☯ These authors contributed equally to this work.

Introduction

In the face of growing energy costs, dwindling fossil resources, environmental pollution and a globalized economy, the large-scale use of biotechnology instead of, or to complement, traditional industrial production processes, particularly in the chemical sector, is viewed as both an opportunity and a necessity to a more social and ecological sustainable energetic matrix [1]. In this context, metagenomics has received much attention owing to its great potential to provide new biocatalysts with diverse functions and applications. To illustrate that, a soil sample might contain in the order of 104_{different bacterial species and more than one} million novel open reading frames, many of which encode

putative enzymes [1]. This approach has been broadly employed to find new enzymes to assist ethanol production through lignocellulosic biomass degradation thereby enabling a new generation of biofuels production [2].

The current scenario for the reduction of biomass into fermentable sugars via enzymatic routes is characterized by very scanty exemplars of glycoside hydrolases suitable for this process in an industrial scale and rate. Thus, the discovery of new enzymes with higher catalytic efficiency and stability under industrial conditions, and even specialized for the different biomass sources, such as sugar-cane bagasse, may have a revolutionary role in making biofuel production from plant biomass economically viable [3,4]. It is more pronounced when focused on cellulose degradation since cellulases are

164

Artigo 5

Development and Biotechnological Application of a

Novel Endoxylanase Family GH10 Identified from

Sugarcane Soil Metagenome

Thabata M. Alvarez1,2._{, Rosana Goldbeck}1._{, Camila Ramos dos Santos}3._{, Douglas A. A. Paixa˜o}1_,

Thiago A. Gonc¸alves1,2_{, Joa˜o Paulo L. Franco Cairo}1,2_{, Rodrigo Ferreira Almeida}1_{, Isabela de Oliveira}

Pereira1_{, George Jackson}1_{, Junio Cota}1_{, Fernanda Bu¨chli}1,2_{, Ana Paula Citadini}1_{, Roberto Ruller}1_{, Carla}

Cristina Polo3_{, Mario de Oliveira Neto}4_{, Ma´rio T. Murakami}3_{*, Fabio M. Squina}1_*

1 Laborato´rio Nacional de Cieˆncia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Saõ Paulo, Brasil, 2 Departamento de Bioquı´mica, Instituto de Biologia (IB), Universidade Estadual de Campinas (UNICAMP), Campinas, Saõ Paulo, Brasil, 3 Laborato´rio Nacional de Biocieˆncias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Saõ Paulo, Brasil, 4 Departamento de Fı´sica e Biofı´sica, Instituto de Biocieˆncias, Universidade Estadual Paulista (UNESP), Botucatu, Saõ Paulo, Brasil

Abstract

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45uC. The crystal structure was solved at 2.75 A˚ resolution, revealing the classical (b/a)8-barrel fold with a conserved active-

site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the+1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.

Citation: Alvarez TM, Goldbeck R, Santos CRd, Paixa˜o DAA, Gonc¸alves TA, et al. (2013) Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome. PLoS ONE 8(7): e70014. doi:10.1371/journal.pone.0070014

Editor: Mark R. Liles, Auburn University, United States of America Received April 22, 2013; Accepted June 13, 2013; Published July 29, 2013

Copyright: ! 2013 Alvarez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was financially supported by FAPESP: 2008/58037-9 (FMS), 2010/11469-1 (TMA), 2012/18559-5 (RG), 2011/20977-3 (JPLFC) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist * E-mail: fabio.squina@bioetanol.org.br (FMS); mario.murakami@lnbio.cnpem.br (MTM) . These authors contributed equally to this work.

Introduction

The conversion of lignocellulosic biomass into renewable fuels has been considered a promising technology to replace fossil fuels and to attend the global need for clean energy. Although the recent worldwide growth in the production of biofuels from plant biomass, several technological bottlenecks still exist and this bioconversion process is not profitable yet [1]. The development of low cost biocatalysts and the improvement of the catalytic efficiency are considered the key to the future of biofuels [1,2,3,4]. Xylanases (E.C. 3.2.1.8) are hemicellulases responsible for breaking down xylan, the major hemicellulosic component of plant cell walls, into short xylooligosaccharides by a general acid–base mechanism involving two glutamic acid residues [5,6]. Typically, these enzymes can be classified into glycoside hydrolase (GH) families 10 and 11 based on amino-acid sequence similarities [7]. Recently, these enzymes have received much attention owing to

their use in degradation of lignocellulosic biomass for biofuels production [8,9].

The development of novel enzymes is primarily dependent on the effectiveness of the screening strategy and the variety of candidate microorganisms present in certain environmental location [10]. In addition, less than 1% of microorganisms from natural environments can be cultivated using traditional culture methods [11]. Thus, researchers have developed strategies to prospect genes through culture-independent methods [1]. Meta- genomics is an advanced strategy to seek for novel functional genes and/or biologically active compounds, by means of direct extraction of all microbial genomic DNA from an environmental

No documento Investigação de vias de degradação de biomassa lignocelulósica no cupim Coptotermes gestroi complementares a hidrolases glicosídicas visando aplicação na produção de bioprodutos : Investigation of lignocellulosic degradation by the termite Coptotermes ges (páginas 159-171)