This thesis describes a new approach to the rational design of an artificial esterase, and is in three parts. The first part of the thesis is an introduction to the design and synthesis of artificial enzymes. This details both traditional 'design', and more recent 'selection' approaches to artificial enzymes, and discusses the advantages and problems associated with the different strategies. The second part is a discussion of our investigations into the design and synthesis of polymeric artificial enzymes which are able to catalyse an ester hydrolysis. The research uses a novel strategy that combines the 'design' and 'selection' approaches: Using the concept of transition state theory, we aimed to design a unit that should bind and stabilise the transition state of a reaction. We then aimed to selectively incorporate this, and other catalytically active groups into a flexible, soluble polymer. The work starts with the synthesis of the ester substrate, and a phosphonate transition state analogue. The 'design' section of the project involved finding a low molecular weight 'binding unit' that could bind to the transition state analogue, and hence should bind and stabilise the transition state of our reaction. We decided to use dipeptides and studied binding using Pulsed Field Gradient NMR techniques to measure changes in diffusion coefficients. This is a technique which had previously only been used to study large molecule-small molecule binding. We successfully managed to apply this technique to studying small molecule-small molecule interactions, and thus it was found that the dipeptide arginine-arginine bound well to our transition state analogue, and the nature of the interactions were studied using molecular modelling. The 'selection' section involved incorporation of this unit into a polymer, along with the introduction of amino acids that could act as catalytically active groups. Both the dipeptide, and chosen amino acids were reacted with a polyallylamine backbone using standard peptide chemistry. The influences of the different polymers on the hydrolysis of the ester were investigated, and it was found that some of these polymers showed distinct catalytic 'enzyme like' properties. The results are reported within. The third section described the experimental work and procedure used throughout this thesis.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-08-31T16:06:08Z No. of bitstreams: 1 texto completo.pdf: 1641093 bytes, checksum: f6c2e8cb821012534ecca293ccb32197 (MD5) / Made available in DSpace on 2017-08-31T16:06:08Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1641093 bytes, checksum: f6c2e8cb821012534ecca293ccb32197 (MD5) Previous issue date: 2017-03-01 / A cenoura (Daucus carota) é cultivada em várias regiões do Brasil, incluindo Minas Gerais. O estado possui três pólos principais de produção: Alto Paranaíba, Triângulo Mineiro e Campos das Vertentes. O nematoide-de-galhas do gênero Meloidogyne está entre os fatores mais importantes na redução de produção de diversas culturas, e a identificação acurada desses fitopatógenos é fundamental para o sucesso das práticas de manejo. Diferentes metodologias são usadas para a diagnose do nematoide-de-galhas. A análise do padrão perineal de fêmeas foi a principal técnica de identificação de espécies de Meloidogyne por vários anos, sendo complementada, a partir da década de 70 e 80, pela técnica de eletroforese de isoenzimas. Atualmente, as técnicas moleculares vêm sendo cada vez mais usadas para diagnose desses patógenos, com diversos protocolos e sequências de DNA disponíveis. Apesar da importância do nematoide-de-galhas para a cultura da cenoura, não há nenhum levantamento sistemático de ocorrência e distribuição do patógeno nessa cultura no estado de Minas Gerais. Mediante ao exposto, o presente trabalho objetivou caracterizar, através de técnicas morfológicas, bioquímicas e moleculares, as principais espécies de Meloidogyne presentes em áreas de cultivo de cenoura em Minas Gerais. Amostras de raízes e solo foram coletadas em áreas infestadas por Meloidogyne spp. na regiões do Alto Paranaíba, Triângulo Mineiro e Campo das Vertentes. A identificação das espécies foi realizada por análise do padrão perineal, eletroforese de isoenzimas e por PCR com primers SCAR específicos. As espécies encontradas foram Meloidogyne incognita, Meloidogyne javanica e Meloidogyne polycephannulata. M. incognita foi a espécie mais frequente, ocorrendo em 9 amostras (60%), seguida de M. javanica (26,6%). M. incognita foi identificado em amostras provenientes das três regiões, enquanto M. javanica em amostras do Alto Paranaíba e Campo das Vertentes. No Alto Paranaíba foi identificada também uma população de Meloidogyne polycephannulata. / Carrots (Daucus carota) are grown in different regions of Brazil, including Minas Gerais. The state has three main production poles: Alto Paranaíba, Triângulo Mineiro and Campos das Vertentes. The root-knott nematode of the genus Meloidogyne is among the most important factors in the reduction of production of several crops, and the accurate identification of these phytopathogen is fundamental for the success of the management practices. Different methodologies are used for the diagnosis of the root-knott nematode. The analysis of the perineal pattern of females was the main technique of identification of Meloidogyne species for several years, being complemented, from the decade of 70 and 80, by the technique of electrophoresis of isoenzymes. Currently, molecular techniques have been increasingly used for the diagnosis of these pathogens, with several protocols and available DNA sequences. Despite the importance of root-knott nematode for carrot culture, there is no systematic study of the occurrence and distribution of the pathogen in this crop in the state of Minas Gerais. The present work aimed to characterize, through morphological, biochemical and molecular techniques, the main species of Meloidogyne present in areas of carrot cultivation in Minas Gerais. Root and soil samples were collected in areas infested by Meloidogyne spp. in the regions of Alto Paranaíba, Triângulo Mineiro and Campo das Vertentes. The identification of the species was performed by perineal pattern analysis, isoenzyme electrophoresis and by PCR with specific SCAR primers. The species found were Meloidogyne incognita, Meloidogyne javanica and Meloidogyne polycephannulata. M. incognita was the most frequent species, occurring in 9 samples (60%), followed by M. javanica (26.6%). M. incognita was identified in samples from the three regions, while M. javanica in samples from Alto Paranaíba and Campo das Vertentes. In Alto Paranaíba a population of Meloidogyne polycephannulata was also identified.

SILVA, M. C. L. Identificação e caracterização de espécies de Meloidogyne em áreas agrícolas e dispersão de M. enterolobii em pomares de goiabeiras no estado do Ceará. 2014. 107 F. Tese (Doutorado em Agronomia/Fitotecnia) - Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, 2014. / Submitted by José Jairo Viana de Sousa (jairo@ufc.br) on 2015-06-11T21:07:41Z No. of bitstreams: 1 2013_tese_mclsilva.pdf: 3677775 bytes, checksum: dc6eca46c746a2f798aa9281e87b5b1a (MD5) / Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2015-06-11T21:08:33Z (GMT) No. of bitstreams: 1 2013_tese_mclsilva.pdf: 3677775 bytes, checksum: dc6eca46c746a2f798aa9281e87b5b1a (MD5) / Made available in DSpace on 2015-06-11T21:08:33Z (GMT). No. of bitstreams: 1 2013_tese_mclsilva.pdf: 3677775 bytes, checksum: dc6eca46c746a2f798aa9281e87b5b1a (MD5) Previous issue date: 2014 / Os nematoides pertencentes ao gênero Meloidogyne estão entre os maiores agentes causadores de danos em plantas, pois possuem ampla distribuição geográfica e são de difícil controle. Considerando que as informações atualizadas sobre as espécies de Meloidogyne afetando plantas em áreas de produção agrícola ainda são escassas no Estado do Ceará, conduziu-se a presente pesquisa com os seguintes objetivos: identificar espécies e raças de Meloidogyne que ocorrem nas diferentes microrregiões do Estado do Ceará e identificar hospedeiras de M. enterolobii em pomares de goiabeira. Cento e doze amostras de plantas infestadas foram coletadas em 29 municípios em áreas produtoras do estado pertencentes a 13 diferentes microrregiões. Verificou-se que das 112 populações obtidas nas coletas, 46 apresentaram fenótipos típicos de M. enterolobii, 27 de M. incognita, 15 de M. javanica, cinco de M. arenaria, uma de M. hapla. Seis populações apresentaram fenótipos de esterase distintos daqueles conhecidos para as espécies de Meloidogyne já relatadas no Brasil. Na determinação das raças fisiológicas, foram encontradas as raças 2 e 3 para M. incognita, a raça 1 para M. arenaria e a raça 2 para M. javanica. As seguintes associações encontradas neste trabalho não foram ainda mencionadas podendo ser os primeiros relatos no estado: M. incognita em acelga (Beta vulgaris var. cicla L.), beterraba (B. vulgaris L.), pimenta de cheiro (Capsicum chinensi Jacq.) e umbu-cajá (Spondia tuberosa x S. mombim); M. javanica em botão de ouro (Siegesbeckia orientalis L.), cajá (S. mombim L.), papaconha (Hybanthus ipecacuanha (L.) Oken.) e quiabo (Abelmoschus esculentus L.); M. arenaria em maria-sem-vergonha (Impatiens walleriana L.), noni (Morinda citrifolia L.) e pingo de ouro (Duranta repens L. var. aurea); M. hapla em roseira (Rosa sp.). A espécie M. enterolobii foi identificada em todas as amostras de raízes de goiabeira coletadas em pomares distribuídos em 13 municípios do estado. Além da goiabaeira, M. enterolobii foi constatada também em acerola (Malpighia glabra L.), batata doce (Ipomoea batatas (L.) Lam), berinjela (Solanum melongena L.), cactos (Cactus sp.), falsa serralha (Emilia fosbergii Nicolson), Hypericum sp, ingá (Inga edulis Mart.), mamão (Carica papaya L.), manjericão (Ocimum basilicum L.), maria-pretinha (Solanum americanum Mill), jurubeba (S. paniculatum L.), palma (Gladiolus sp.) e pimenta tabasco (C. frutescens L.), associações estas relatadas pela primeira vez no Estado do Ceará. A espécie M. enterolboii estava presente em 52% dos municípios e em 77% das microrregiões visitadas enquanto que M. incognita e M. javanica foram constatadas em 35% e 31% dos municípios, respectivamente, ambas em 46% das microrregiões visitadas. Este estudo vem contribuir na atualização das informações sobre a ocorrência de espécies de Meloidogyne nas principais regiões produtoras do Estado do Ceará. / The nematodes from the genus Meloidogyne are the most important causal agents of plant damages considering that they have a large geographical distribution and they are difficult to be controlled. Considering that the actual information about species from the genus Meloidogyne infecting plants in agriculture areas are still short in the State of Ceará, the present research was developed with the following objectives: identify Meloidogyne species and races which occur in the different micro-regions from the State of Ceará and identify the natural hosts for M. enterolobii in guava (Pisidium guajava) orchards. A hundred and twelve plants with gall symptoms in the roots were collected from producing areas of 29 counties including 13 micro-regions from the State of Ceará. It was observed that 112 nematode populations collected from infected plants, 46 presented phenotype typical of M. enterolobii, 27 of M. incognita, 15 of M. javanica, five of M. arenaria and one of M. hapla. Six nematode populations presented esterase phenotype distinct from those known for the Meloidogyne species already described in Brazil. In the physiologic race determination it was found races 2 and 3 of M. incognita, and race 1 of M. arenaria and a race 2 of M. javanica. The following nematode associations found in the present paper were not yet mentioned which could demonstrate that they could be the first report in the State: M. incognita in Beta vulgaris var. cicla L., B. vulgaris L., Capsicum chinensi Jacq. and Spondia tuberosa x S. mombim; M. javanica in Sieges beckiaorientalis L., S. mombim L., Hybanthus ipecacuanha (L.) Oken. and Abelmoschus esculentus L.; M. arenaria in Impatiens walleriana L., Morinda citrifolia L. and Duranta repens L. var. aurea; and M. hapla in Rosa sp. Meloidogyne enterolobii was identified in all guava root samples collected in the orchards distributed in 13 counties from the State. Besides guava, M. enterolobii was also detected in Malpighia glabra L., Ipomoea batatas (L.) Lam, Solanummelongena L., Cactus sp., Emilia fosbergii Nicolson, Hypericum sp., Inga edulis Mart., Carica papaya L., Ocimum basilicum L., Solanum americanum Mill, S. paniculatum L., Gladiolus sp. and C. frutescens L. The present research is the first report about those plant nematode associations in the State of Ceará. The specie M. enterolboii was present in 52% of the counties and in 77% of the micro-regions visited, while M. incognita and M. javanica were detected only in 35% and 31% of the visited counties, respectively, and in 46% of the micro-region visited. The present research will contribute to update the scientific information about the occurrence of Meloidogyne species in the main agriculture producing regions from the State of Ceará.

O comércio mundial de enzimas industriais estava estimado em 2.3 bilhões de dólares entre detergentes (U$ 789 milhões), aplicações alimentícias (U$ 634 milhões), agricultura (U$ 237 milhões), entre outros. Neste contexto, as enzimas lipolíticas estão atraindo enorme atenção devido ao seu potencial biotecnológico, visto que estas podem catalisar múltiplas reações (hidrólise, acidólise, interesterificação e glicerólise). Enzimas lipolíticas de origem microbiana são economicamente atrativas por serem biodegradáveis, atuarem normalmente em condições brandas, e serem quimio-seletivas propiciando à indústria farmacêutica a obtenção de drogas com efeito colateral reduzido. Neste projeto, quatro genes potenciais codificadores de esterases/lipases, advindos de uma biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel, foram clonados em vetores de expressão e expressos em Escherichia coli BL21 (DE3), e as proteínas correspondentes foram submetidas a ensaios funcionais e estruturais. / The global trade of industrial enzymes is estimated at 2.3 billion U.S. dollars, divided mainly between detergents (US$ 789 million), food applications (US$ 634 million), and agriculture (US$ 237 million). Within this trade, lipolytic enzymes have attracted enormous attention because of their biotechnological potential as catalysts of multiple reaction types (including hydrolysis, acidolysis, interesterification and glycerolysis). Lipolytic enzymes of microbial origin are economically attractive because they are easily biodegradable, usually act in mild conditions, and are chemo-selective, providing the pharmaceutical industry a method for obtaining drugs with reduced side effects. In this project, four individual genes encoding putative esterases/lipases identified in a metagenomic library obtained from a microbe consortium isolated from diesel oil-contaminated soil were cloned into expression vectors and expressed in Escherichia coli BL21 (DE3), and their corresponding recombinant proteins were used for functional and structural studies.

O comércio mundial de enzimas industriais estava estimado em 2.3 bilhões de dólares entre detergentes (U$ 789 milhões), aplicações alimentícias (U$ 634 milhões), agricultura (U$ 237 milhões), entre outros. Neste contexto, as enzimas lipolíticas estão atraindo enorme atenção devido ao seu potencial biotecnológico, visto que estas podem catalisar múltiplas reações (hidrólise, acidólise, interesterificação e glicerólise). Enzimas lipolíticas de origem microbiana são economicamente atrativas por serem biodegradáveis, atuarem normalmente em condições brandas, e serem quimio-seletivas propiciando à indústria farmacêutica a obtenção de drogas com efeito colateral reduzido. Neste projeto, quatro genes potenciais codificadores de esterases/lipases, advindos de uma biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel, foram clonados em vetores de expressão e expressos em Escherichia coli BL21 (DE3), e as proteínas correspondentes foram submetidas a ensaios funcionais e estruturais. / The global trade of industrial enzymes is estimated at 2.3 billion U.S. dollars, divided mainly between detergents (US$ 789 million), food applications (US$ 634 million), and agriculture (US$ 237 million). Within this trade, lipolytic enzymes have attracted enormous attention because of their biotechnological potential as catalysts of multiple reaction types (including hydrolysis, acidolysis, interesterification and glycerolysis). Lipolytic enzymes of microbial origin are economically attractive because they are easily biodegradable, usually act in mild conditions, and are chemo-selective, providing the pharmaceutical industry a method for obtaining drugs with reduced side effects. In this project, four individual genes encoding putative esterases/lipases identified in a metagenomic library obtained from a microbe consortium isolated from diesel oil-contaminated soil were cloned into expression vectors and expressed in Escherichia coli BL21 (DE3), and their corresponding recombinant proteins were used for functional and structural studies.

A parede celular das plantas inclui componentes polissacarídeos complexos, e a sacarificação destes polímeros necessita da ação de conjuntos de enzimas que atuem em sinergia. Enzimas podem formar complexos multi-enzimáticos que possuem mais de uma atividade catalítica derivada de domínios distintos de uma mesma cadeia polipeptídica. O objetivo deste trabalho foi construir uma enzima bifuncional com os domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) para desconstrução de material lignocelulósico de cana-de-açúcar. Para isso, dois diferentes domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) oriundos de Clostridium thermocellum foram fundidas para criar a quimera feruloil esterase/acetil-xilano esterase (FaeAxe). O desenho racional da quimera foi feito utilizando-se de métodos computacionais, que permitiram a criação de um modelo estrutural da enzima. A construção da quimera foi feita por overlap PCR, clonada em vetor pET-SUMO e expressa em Escherichia coli. As duas enzimas parentais (Fae e Axe) foram clonadas em vetor pET28 e expressas em E. coli. Durante a etapa de expressão, observou-se que todas as enzimas foram expressas na forma solúvel. As enzimas feruloil esterase e acetilxilano esterase têm como substrato o polímero arabinoxilano, cuja degradação é uma etapa chave na sacarificação de biomassa. Dessa forma, as atividades da quimera, bem como das enzimas parentais foram testadas contra polímeros arabinoxilano de trigo e arabinoxilano de cana-de-açúcar após a hidrólise pela endoxilanase GH11 de Bacillus Subtilis e analisadas por meio de espectrometria de massas. A atividade desacetilase da enzima parental acetil-xilano esterase e da quimera FaeAxe foram confirmadas, evidenciando que a quimera preservou essa atividade catalítica. A atividade da enzima feruloil esterase e da quimera FaeAxe na remoção de ácido ferúlico dos oligossacarídeos gerados pela endoxilanase GH11 não foi observada / The plant cell wall is comprised of a matrix of polysaccharides and saccharification of these polymers requires the joint action of diverse enzymes. Enzymes may form multi-enzymatic complexes that have more than one catalytic activity derived from different domains of a single polypeptide chain. The aim of this work was to construct a bifunctional enzyme with two catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) for degradation of sugar cane lignocellulosic material. The two different catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) from Clostridium thermocellum were fused to generate a bifunctional chimera feruloyl esterase/acetylxylan esterase (FaeAxe). A molecular model was created by rational design using a 3D-structure guided strategy. The fusion was created using overlap PCR, and the resulting product was cloned into the pETSUMO vector. The chimeric protein and the parental enzymes were expressed in Escherichia coli and purified and the enzymes were expressed in soluble form. Xylanases, feruloyl esterases and acetylxylan esterases degrade arabinoxylan polymers and their activity is a key step in the saccharification of biomass. The catalytic properties of the chimera and of the parental enzymes were tested against wheat and sugarcane arabinoxylan polymers after hydrolysis by GH11 endoxylanase from Bacillus subtilis and analyzed by mass spectroscopy. The deacetylase activity of acetyl-xylan esterase parental enzyme and FaeAxe chimera were confirmed, showing that the chimera kept the deacetylase activity. After hydrolysis by GH11 endoxylanase from Bacillus subtilis the feruloyl esterase and FaeAxe chimera activities on ferulic acid removal were not observed

Reaction rates and selectivities were measured for transacylation of fatty acid esters in solvents catalysed by Candida antarctica lipase B and by cutinase from Humicola insolens. With these enzymes classical water-based enzymology can be expanded to many different solvents allowing large variations in interaction energies between the enzymes, the substrates and the surrounding. Further ,hydrolysis reactions catalysed by Bacillus subtilis esterase 2 were investigated. Thermodynamics analyses revealed that the enzyme contribution to reaction rate acceleration compared to acid catalysis was purely entropic. On the other hand, studies of differences in activation entropy and enthalpy between enantiomers and between homologous esters showed that high substrate specificity was favoured by enthalpic stabilisation. Solvent was found to have a profound effect on enzyme catalysis, affecting both reaction rate and selectivity. Differences in substrate solubility will impact enzyme specificity since substrate binding is an equilibrium between enzyme-bound substrate and substrate in free solution. In addition, solven tmolecules were found to act as enzyme inhibitors, showing both competitive and non-competitive behaviour. In several homologous data series enthalpy-entropy compensation relationships were encountered. A possible extrathermodynamic relationship between enthalpy and entropy can easily be lost under co-varying errors propagated from the experiments. From the data in this thesis, one instance was found of a real enthalpy-entropy compensation that could be distinguished from statistical errors, while other examples could not be verified. / QC 20100722

Thesis--Leyden. / In Periodical Room.

Nonspecific esterase, and in some cases a "Tween" lipase, were investigated histochemically in arteries and several other tissues of rats and rabbits under various conditions known to affect the development of atherosclerosis: age and sex differences, endocrine and metabolic factors (sex steroids, thyroid function, alloxan diabetes, and fasting), arterial injury, and acute and chronic lipemia. A study of the above enzymes was also made in atherosclerotic rabbit and human aortae. Of the several nonvascular tissues studied, only the enzymes of the male gonads and a male accessory sex organ (the prostate) were influenced by some of the experimental conditions tested: maturation greatly increased the activity of both enzymes in the Leydig cells of the testis, and stilbestrol markedly diminished the esterase activity in the prostate epithelium. Thus, in certain reproductive organs, the enzymes reflected the functional state of the cells under investigation. The following findings were made concerning the enzymes of the arterial wall: 1. Neither of the enzymes was influenced by ageing or sex difference in the rat, rabbit or human. 2. Neither of the enzymes was influenced by sex hormones, acute or chronic lipemia, or any of the metabolic conditions studied in the rat. 3. Esterase disappeared from the foci of acute arterial injury induced by Calciferol treatment in the rat and did not reappear six weeks after infliction of the injury. 4. Strong esterase activity appeared in the cytoplasm of lipo-phages within both human and experimental rabbit atherosclerotic lesions, in contrast to the normal arterial wall which exhibited no esterase activity at all in either species with the methods used. 5. Esterase was absent from the superficial fibrous cap of the human atherosclerotic lesion. It is concluded that: In the rat arteries, which normally exhibit appreciable esterolytic activity, this enzyme appears to be quite stable since it is not visibly modified by ageing or a series of endocrine and metabolic influences; it is, however, drastically diminished by acute vascular injury and this may account, at least in part, for the well known preferential accumulation of lipids in foci of acute arterial damage. In the rabbit arteries, which normally exhibit no histochemically demonstrable esterase, appreciable esterolytic activity appears only within the cytoplasm of cells that have taken up lipid after exposure to chromic lipemia (foam cells of atheromata). Similarly, in human atheromata esterase appeared only in foam cells and was absent from the fibrous cap of the atherosclerotic lesions; thus the absence of lipid from the cap cannot be attributed to increased enzyme activity in this part of the lesion. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

A parede celular das plantas inclui componentes polissacarídeos complexos, e a sacarificação destes polímeros necessita da ação de conjuntos de enzimas que atuem em sinergia. Enzimas podem formar complexos multi-enzimáticos que possuem mais de uma atividade catalítica derivada de domínios distintos de uma mesma cadeia polipeptídica. O objetivo deste trabalho foi construir uma enzima bifuncional com os domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) para desconstrução de material lignocelulósico de cana-de-açúcar. Para isso, dois diferentes domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) oriundos de Clostridium thermocellum foram fundidas para criar a quimera feruloil esterase/acetil-xilano esterase (FaeAxe). O desenho racional da quimera foi feito utilizando-se de métodos computacionais, que permitiram a criação de um modelo estrutural da enzima. A construção da quimera foi feita por overlap PCR, clonada em vetor pET-SUMO e expressa em Escherichia coli. As duas enzimas parentais (Fae e Axe) foram clonadas em vetor pET28 e expressas em E. coli. Durante a etapa de expressão, observou-se que todas as enzimas foram expressas na forma solúvel. As enzimas feruloil esterase e acetilxilano esterase têm como substrato o polímero arabinoxilano, cuja degradação é uma etapa chave na sacarificação de biomassa. Dessa forma, as atividades da quimera, bem como das enzimas parentais foram testadas contra polímeros arabinoxilano de trigo e arabinoxilano de cana-de-açúcar após a hidrólise pela endoxilanase GH11 de Bacillus Subtilis e analisadas por meio de espectrometria de massas. A atividade desacetilase da enzima parental acetil-xilano esterase e da quimera FaeAxe foram confirmadas, evidenciando que a quimera preservou essa atividade catalítica. A atividade da enzima feruloil esterase e da quimera FaeAxe na remoção de ácido ferúlico dos oligossacarídeos gerados pela endoxilanase GH11 não foi observada / The plant cell wall is comprised of a matrix of polysaccharides and saccharification of these polymers requires the joint action of diverse enzymes. Enzymes may form multi-enzymatic complexes that have more than one catalytic activity derived from different domains of a single polypeptide chain. The aim of this work was to construct a bifunctional enzyme with two catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) for degradation of sugar cane lignocellulosic material. The two different catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) from Clostridium thermocellum were fused to generate a bifunctional chimera feruloyl esterase/acetylxylan esterase (FaeAxe). A molecular model was created by rational design using a 3D-structure guided strategy. The fusion was created using overlap PCR, and the resulting product was cloned into the pETSUMO vector. The chimeric protein and the parental enzymes were expressed in Escherichia coli and purified and the enzymes were expressed in soluble form. Xylanases, feruloyl esterases and acetylxylan esterases degrade arabinoxylan polymers and their activity is a key step in the saccharification of biomass. The catalytic properties of the chimera and of the parental enzymes were tested against wheat and sugarcane arabinoxylan polymers after hydrolysis by GH11 endoxylanase from Bacillus subtilis and analyzed by mass spectroscopy. The deacetylase activity of acetyl-xylan esterase parental enzyme and FaeAxe chimera were confirmed, showing that the chimera kept the deacetylase activity. After hydrolysis by GH11 endoxylanase from Bacillus subtilis the feruloyl esterase and FaeAxe chimera activities on ferulic acid removal were not observed