Xylanases are xylan-degrading enzymes, belong to glycosyl hydrolases (GH). Xylanases from the two major families 10 (GH10) and 11 (GH11) catalyze the hydrolysis of internal β-, bonds of xylan backbone. Xylan is the second most abundant polysaccharide in nature. Nearly one third of the dry weight of the higher plants is xylan and therefore, xylanases have an important role in biomass conversions. Currently, the most effective application of xylanases is in prebleaching of kraft pulp to minimize the use of environmentally hazardous chemicals in the subsequent treatment stages. In recent years, therefore, attention is focused on to isolate and/or engineer the xylanases for the industrial requirements. The desirable roperties of xylanases in paper industry are stability and activity at high temperatures and alkaline pH. While he factors responsible for the thermal stability of GH10 xylanases have been analyzed, factors governing the alkaline stability of GH10 xylanases remain poorly understood. The present thesis reports the crystal structures of an alkali thermostable GH10 extracellular endo-xylanase (BSX) from an alkalophilic organism, Bacillus sp. NG-27 in free and xylosaccharides-bound form. The enzyme was purified from the native organism and crystallized. The structure was solved by molecular replacement method. The 2.2 Å crystal structure of the native BSX enzyme is the first structure of an alkali thermostable GH10 family xylanase from an alkalophilic organism. It has unveiled unique protein properties that can form the basis for improving the thermal, alkaline stability and activity by site directed mutagenesis. The comparative study, especially in relation to GH10 xylanases, deciphered important structural features which are likely to be responsible for the alkaline stability of the enzyme. The work exemplifies the mechanism of adaptation of enzymes to function under polyextreme conditions through changes in the nature and composition of solvent-exposed residues. As apparent from the comparative study, the enhanced stability of the protein can be attributed to the surface rich in acidic residues and less number of solvent-exposed Asn as seen in BSX. This situation which may be roughly described as “acidic residues outside and Asn inside”, is a notable feature of alkali-stable GH10 xylanases from alkalophilic organisms. In addition, the candidate has carried out the comprehensive database analysis of the occurrence of C-H…O hydrogen bonds in helices and helix termini of globular proteins. The study provides a compelling evidence that the main-chain Cα and the side-chains CH which participate in C-H…O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N-H…O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins. Chapter 1 starts with a brief introduction of xylanases, their classifications and overall folds. At present, a little more than a dozen of crystal structures of GH10 xylanases are known and described in the literature. A brief mention about these structures and their optimum pH and temperature is outlined under a separate section. In view of the industrial importance of the study enzyme, the potential industrial and biotechnological applications of xylanases are detailed in this Chapter. A section is dedicated to describe the present study enzyme BSX, an alkali thermostable endo-xylanase from an alkalophilic bacterium, Bacillus sp. NG-27. BSX has a molecular mass of ~41 kDa and is optimally active at 343 K and at a pH of 8.4. The alkaline thermostability of the wild type BSX is likely to be industrially important. At the end, the scope of the present work is detailed. Chapter 2 presents the purification of xylanase (BSX) from Bacillus sp. NG-27, the crystallization of the native and xylosaccharides-bound BSX, the X-ray diffraction data collection on these crystals and processing of the data. Repeated attempts to crystallize the protein expressed in the chloroplast of transgenic tobacco plant were unsuccessful. However, crystallization was achieved with the protein sample purified from the native source by hanging drop vapour diffusion method. Crystals were grown at both acidic (4.6) and basic pH (8.5). The corresponding crystallization conditions are 0.2 M MgCl2, 0.1 M sodium acetate pH 4.6 and 20% PEG 550 MME and 0.1 M aCl, 0.01 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000. Crystals grown at acidic pH were not suitable for X-ray diffraction study. Subsequently, crystal obtained at a basic pH of 8.5 was used for X-ray data collection and it diffracted X-rays to better than 2.2 Å at the home source at cryo-temperature (100 K). Native BSX crystals belong to monoclinic space group C2 with unit cell parameters a = 174.5 Å, b = 54.7 Å, c = 131.5 Å and β = 131.2°. Crystals of xylosaccharides-bound enzyme were grown in a slightly modified crystallization condition of native, 0.1 M NaCl, 0.2 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000 and the enzyme was incubated with xylan prior to setting up the crystallization. Crystals belong to primitive orthorhombic space group P212121 with unit cell parameters a = 59.2 Å, b = 83.8 Å and c = 174.4 Å. A data set was collected using synchrotron radiation of wave length 1.0 Å from a cryo-cooled crystal at Spring-8 BL26B1 beam line, Japan. The Matthews coefficient VM for native and xylosaccharides- bound crystals was calculated to be 2.8 and 2.7 Å3 Da-1, respectively, suggesting two molecules in each crystal asymmetric unit. No twinning was detected in both the datasets and the overall quality of the data sets was found to be good. Chapter 3 details the application of molecular replacement method to the structure solution of native and xylosaccharides-bound BSX, the course of iterative model building and the refinement carried out, and the quality of the final protein structure models. The native-enzyme structure solution was obtained by the molecular replacement method using as a search model the crystal structure (PDB code 1hiz) of the closest homologous, extracellular xylanase (GSX) from Geobacillus stearothermophilus. No non- crystallographic symmetry (NCS) restraint was applied between the two independent molecules in the crystal asymmetric unit at the final round of refinement. The final positional refinement of native BSX converged to R factors of R = 19.4% and Rfree = 23.5% for data between 20.0 to 2.2 Å. The final native model consists of 5704 protein atoms, two Mg2+ ions and 721 solvent water molecules. The final native model was taken as the search structure for the xylosaccharides-bound BSX and a solution with a correlation coefficient of 70.7% and an R-factor of 32.1% was obtained from the molecular replacement calculation. Unlike the native structure refinement, NCS restraint was imposed at all stages of the refinement. Bound xylosaccharides were clearly visible inthe difference Fourier electron density maps. The last round of refinement gave a model with R and Rfree of 21.8% and 25.7%, respectively. The final xylosaccharides-bound model consists of 5766 protein atoms, four Mg2+ ions, 85 atoms belong to bound xylosaccharides and 523 solvent water molecules. No residues were found in the disallowed region of the Ramachandran (φ, ψ) map for both the structures. Chapter 4 describes the native and xylosaccharides-bound BSX crystal structures and the structural comparison of BSX with other GH10 family xylanase crystal structures for which the optimum temperature and pH are known in the literature. BSX folds as the ubiquitous (β/α)8-barrel, a common structural superfold characteristic of GH10 xylanases. The two active site glutamic acid residues, Glu149 and Glu259, are located on opposite sides of the active site cleft and their side-chains are at a distance of 5.5 Å apart suggesting the enzymatic reaction takes place by the retaining mechanism. From the structural superposition of other xylotriose-bound xylanase structures on to the xylosaccharides-bound BSX, structural plasticity in the xylotriose binding can be inferred, implying that the xylose recognition at the subsite -3 displays plasticity and is less specific as opposed to that at -1 and -2 subsites. The stacking interaction of one of the xylose moieties of the xylobiose with the Trp235 seen in BSX provides, for the first time, a structural evidence for the direct involvement of Trp235 in xylosaccharides binding. The crystal structure revealed a metal binding site, found at the C-terminal end of catalytic domain. The presence of metal binding site was not anticipated from earlier theoretically modeled structure and biochemical studies. Further, we have shown experimentally the requirement of Mg2+ ion for the enzyme activity. We havedescribed a novel WP sequence-structure-interaction motif which is present in the (+) side of the active site region and presumably helps in the efficient binding of the carbohydrate moiety of the xylan in the active site cleft of BSX. The structural comparison of BSX with other GH10 xylanases solved to date and characterized to be active at a pH close to neutral was done for the first time. The comparative study revealed the essential structural features which may responsible for the alkaline stability of GH10 xylanases.Briefly, the alkalophilic GH10 xylanases from alkalophilic organisms have surface abundant in acidic residues, the heat and alkaline susceptible residue Asn depleted on the protein surface and increased number of salt bridges. Our study has unveiled the role of the nature and composition of protein surface amino acids in the adaptation of enzymes to polyextreme conditions. The observations reported in the thesis provide important lessons for engineering alkaline stability in xylanases for industrial applications and in general for the understanding of alkaline stability in related proteins. A comparison of the surface features of the BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in polyextremophilicity of proteins, where understanding structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. Chapter 5 summaries the important findings of the present study from the crystal structural analysis of BSX and its comparison with non-alkalophilic GH10 xylanases. Separate sections are made on conclusions and future prospects for the study on BSX. Chapter 6 describes the comprehensive database analysis of C-H…O hydrogen bond in helices of globular proteins. The C-H…O hydrogen bonds found in helices are predominantly of type 5 → 1 or 4 → 1.Our analysis reveals that the Cγ and Cβ hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic β-branched residue Ile to participate in 5 → 1 C- H…O hydrogen bonds involving methylene Cγ1 atom as donor in α-helices. In addition, C-H…O hydrogen bonds are present along with helix stabilizing salt bridges and to some extent compensate for the side-chain conformational entropy loss. Our analysis highlights that a multitude of local C-H…O hydrogen bondsformed by a variety of amino acid side-chains and Cα hydrogen atoms occur in helices and more so at the helix termini. A majority of the helix favouring residues, Met, Glu, Arg, Lys, Leu and Gln which also have large side-chains with more donatable CH groups, have significant propensity to form side-chain to main-chain C-H…O hydrogen bonds in helix. The large side-chains are marked by their ability to shield from the solvent the polar atoms of the peptide backbone and at the same time participate in weak cohesive C-H…O interactions in the helix. This chapter also details the identification for the first time a novel chain reversal motif stabilized by 1 → 5 Cα-H…O interactions. The importance of these hydrogen bonds with respect to helix stability is discussed in the final section of the chapter. Appendix A details the crystallographic and structural analyses oftwares used for the present thesis work. Appendix B describes, in addition to the crystal structure analysis of BSX, the work carried out by the candidate on a comparative study of a thermostable xylanase from Thermoascus aurantiacus, solved in our laboratory at atomic 1.11 Å (293 K) and ultrahigh 0.89 Å (100 K) resolutions. From the comparison, we have for the first time pointed out the possibility of plasticity of ion pairs in proteins with water molecules mediating some of the alternate arrangements. The αβ-loops are relatively less flexible than the βα-loops. The β-strands are least affected structurally with the increase in temperature. Thus the TIM barrel fold in the study enzyme, though having a single domain, may be dissected into parts based on the relative flexibility and described as having a rigid core constituted by the β-barrel and a less rigid exterior formed by the surrounding α-helices. Appendix C presents the crystallization and the preliminary X-ray characterization work done by the author of the thesis on an alkali thermostable cellulase enzyme from Thermomonospora sp. The protein is an extracellular enzyme with molecular mass of 14.2 kDa and interestingly, has the dual activity for both cellulose and xylan. The primary structure of the enzyme is not known. The enzyme was purified from the source organism and crystallized. A complete diffraction data set was collected and processed to 2.3 Å in an orthorhombic space group P212121. Appendix D contains tables which give details about the analysed 5 → 1 Cα- H…O hydrogen bonds in helices and a novel chain reversal motif with 1 → 5 Cα-H…O hydrogen bonds. Appendix E encloses reprints of publications which have resulted from the work reported in the thesis.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-08Bitstream added on 2014-06-13T20:04:39Z : No. of bitstreams: 1 terrasan_crf_dr_rcla.pdf: 2923287 bytes, checksum: 6f131c106722e1d7349212e5619e3b4f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Xilanases e β-xilosidases são as principais enzimas responsáveis pela degradação da xilana, o segundo principal constituinte da parede celular vegetal. Tanto a produção destas enzimas por micro-organismos, quanto a caracterização bioquímica das mesmas têm sido amplamente estudadas devido às suas inúmeras aplicações biotecnológicas. Neste trabalho, as principais xilanase e β-xilosidase produzidas por uma linhagem de Penicillium janczewskii em meio de cultura líquido foram purificadas por métodos clássicos de purificação de proteínas, sendo, posteriormente, caracterizadas bioquimicamente. A xilanase apresentou atividade ótima em pH 6,0 e a 65 °C e a β-xilosidase em pH 5,0 e a 75 °C. Ambas as enzimas apresentaram características interessantes principalmente com relação à atividade ótima de ambas as enzimas em elevadas temperaturas, a prolongada estabilidade da β-xilosidase a 60 °C, e a estabilidade da xilanase em pH mais alcalinos, considerando-se algumas de suas possíveis aplicações. Visando uma futura aplicação, a produção destas enzimas foi avaliada em cultivos sólidos utilizando-se como substrato o bagaço de malte, resíduo da indústria cervejeira. As melhores condições para produção enzimática foram: utilização da umidade inicial do substrato de 50% fornecida com solução de sais de Vogel, tempo de cultivo de sete dias a 25 °C para produção de xilanases e a 20 °C para produção de b-xilosidases. O material fermentado apresentou aumento no teor protéico, na quantidade de alguns aminoácidos essenciais e ausência de micotoxinas. Ainda com um enfoque ambiental o filtrado de cultura de P. janczewskii, produzido em condições anteriormente selecionadas, foi aplicado no biobranqueamento de uma polpa kraft de eucalipto pré-branqueada com oxigênio, sendo realizados ensaios para seleção da concentração de xilanases e do tempo de reação / Xylanases and β-xylosidases are the main enzymes responsible for the degradation of xylan, the second main constituent of plant cell walls. The production of these enzymes by microorganisms, and their biochemical characterization has been extensively studied due to their wide range of biotechnological applications. In this work, the main xylanase and β-xylosidase produced in liquid cultures by a Penicillium janczewskii strain were purified by classical methods of protein purification, and further biochemically characterized. The xylanase showed optimal activity at pH 6.0 and 65 °C and β-xylosidases at pH 5.0 and 75 °C. Considering some possibilities of applications, the enzymes presented interesting characteristics especially in relation to the optimal activity at high temperatures, the prolonged stability of the β-xylosidase at 60 °C, and the stability of the xylanase in alkaline pH. Aiming at a future application, the production of these enzymes was investigated in solid state fermentation using brewer’s spent grain, a residue of the brewing industry, as substrate. The optimized conditions were: 50% initial substrate moisture supplied by Vogel’s salt solution, culturing for 7 days at 25 °C for xylanase production and at 20 °C for b-xylosidase production. The fermented substrate showed increase in protein content, in the amount of some essential amino acids, and absence of mycotoxins. Maintaining the environmental focus, the P. janczewskii crude filtrate, produced under previously selected conditions, was applied in the biobleaching of eucalyptus kraft pulp pre-bleached with oxygen. Trials were conducted for the selection of xylanase concentration and reaction time

Master of Science / Food Science Institute / Fadi Aramouni / β-(1,4)-endoxylanases, commonly referred to as xylanases, have become integral to the industrial breadmaking process. This enzyme is known to cause improvement in dough rheology, loaf volume, and crumb grain. Significant research has been conducted regarding the structure, function, and inhibition of xylanases, but there is currently no quick and reproducible method to evaluate their effect in baking. The goal of this research was to develop a quantitative method for this purpose and to determine why the effect of xylanases varies with different wheat flours. The currently used methods of test baking, dough stickiness, and spectrophotometric analysis for reducing sugars were evaluated, and failed to provide reproducible results. Therefore, a new method was developed to measure the Flour Water Expression Rate (FWER) with the addition of xylanases. Commercially available enzymes from Aspergillus niger and Bacillus subtilis were evaluated in this study. The FWER method measures the amount of water released by the xylanase over a set period of time. This method consistently provided statistically significant data (p<0.05), which was able to provide a comparison of xylanases from A. niger and B. subtilis in different flours. The results indicated that the xylanase from A. niger tends to release more water, have a higher FWER value, than the xylanase from B. subtilis. In one flour, A. niger xylanase resulted in an FWER of 15.18 compared to B. subtilis xylanase that resulted in an FWER of 9.57 at equivalent activities. However, inhibitors in the wheat appeared to cause an impact on the FWER, which was evaluated with an uninhibited xylanase from B. subtilis. This new method for the evaluation of xylanases in baking suggests varying levels of xylanase inhibitors in wheat may be the reason xylanases effect wheat flours differently.

Made available in DSpace on 2014-06-11T19:27:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-28Bitstream added on 2014-06-13T19:56:06Z : No. of bitstreams: 1 simoes_mlg_me_rcla.pdf: 924529 bytes, checksum: 4fd62ec448289172b02333ca84efaa63 (MD5) / Neste estudo, foram isoladas 67 linhagens de fungos filamentosos de solo de área de caatinga, sendo as coletas efetuadas em períodos seco e chuvoso, com o objetivo de se conhecer a biodiversidade deste bioma não explorado e avaliar o potencial destes fungos na produção de xilanase. Algumas linhagens não foram identificadas por inexistência de metodologias específicas e outras foram identificadas através de métodos microscópicos e bioquímicos. Foi efetuada uma triagem dos fungos potencialmente produtores desta enzima em meio de Vogel contendo xilano 1% como única fonte de carbono e avaliou-se através do Método Turbidimétrico Automatizado (Bioscreen-C), a melhor fonte de carbono para crescimento dos fungos selecionados. Os resultados obtidos nos cultivos em MFS, foram inexpressivos quando comparados aos obtidos em MFT. Os melhores produtores de xilanase em MFT foram cultivados em meio líquido de Vogel e MFT com adição individual de outras fontes de carbono a 1% (carbometilcelulose- CMC, xilano e a melhor fonte de carbono para crescimento, determinada pelo Bioscreen-C) em temperaturas apropriadas, pH 5, por 5 dias, inóculo de 1 x 107 esporos/mL. O CMC causou repressão catabólica na síntese de xilanase por estes fungos tendo a adição do xilano apresentado o mesmo efeito, com exceção de Trichoderma viride, que teve sua atividade aumentada para 143,0 U/mL e Aspergillus niger 11 que teve sua atividade aumentada de 33,4 U/mL para 57,1 U/mL. / In this study, 67 strains of filamentous fungi were isolated from caatinga area, the collections were performed during the dry and rainy period, aiming to know the biodiversity of this not explored bioma and to evaluate the potential of these fungi to produce xylanase. Some of the strains were not identified due to the lack of specific methodologies and others were identified through microscopic and biochemical methods. A selection of the fungi which were considered potentially producers of xylanase was carried out in Voguel medium containing 1% of xylan as an only carbon source, and through a Automatized Turbidimetric Method (Bioscreen - C) the best carbon source for the fungi growth was evaluated. The results obtained in SBM were not expressive when compared to the ones obtained in WBM. The best xylanase producers in WBM were cultivated in Voguel liquid medium and WBM adding individually other carbon sources at 1% (carboxymethylcellulose -CMC; xylan; and the best growth carbon source determined by Bioscreen-C) at appropriated temperatures, pH 5,0 ; for 5 days, and a spore concentration of 1 x107 spores/mL. The CMC addition caused catabolic repression in xylanase production by these fungi, xylan addition showed the same effect, but Trichoderma viride 13 and Aspergillus niger 11 which have their activities increased from 39,21 U/mL to 143,0 U/mL and from 33,4 U/mL to 57,1 U/mL, respectively.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13347号 / 論農博第2890号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5254(農学部図書室) / (主査)教授 杉山 淳司, 教授 髙部 圭司, 教授 渡邊 隆司 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The overall objective of this study was to evaluate the effects of feeding hulless barley and supplementing a xylanase enzyme on production performance and nutrient utilization of lactating dairy cows. In study 1, we evaluated production performance, milk fatty acid composition, and nutrient digestibility in high-producing dairy cows consuming diets containing corn and hulless barley in different proportions as the grain source. We hypothesized that a plausible reduction in production performance would be explained by an altered rumen function, which would be reflected in a reduction of the proportion of de novo fatty acids in milk fat. The inclusion of hulless barley grain as the energy source in diets for lactating dairy cows resulted in similar production performance and nutrient utilization as corn grain. We concluded that hulless barley is as good as corn grain as an energy source and increasing NDF concentration in hulless barley-based diet is not necessary. In study 2, we evaluated production performance, nutrient digestibility, and milk fatty acid composition of high-producing dairy cows consuming diets containing hulled or hulless barley as the grain source. We hypothesized that rumen function is altered when cows are fed low-forage diets containing barley grains, and this altered rumen function would be reflected in lower production performance and a reduction of fatty acids synthesis in the mammary gland. Contrary to our expectations, feeding hulled barley or hulless barely based diets with different forage to concentrate ratios to lactating dairy cows resulted in similar production performance and nutrient utilization. We concluded that both hulled or hulless barley grains are good energy sources for sustaining high milk production and there is no need to increase NDF concentration in diet when using barley grain as the grain source. In study 3, we evaluated the effects of supplementing a xylanase enzyme on production performance and nutrient digestibility of lactating dairy cows fed diets containing corn or sorghum silage as the forage source. We hypothesized that supplementing a xylanase enzyme product in diets containing corn or sorghum silage increases NDF digestibility, and production performance of lactating dairy cows would also be improved due to enhanced fiber digestion. Supplementation of xylanase for 19 d did not affect cow performance and nutrient utilization. Supplementation of xylanase may require a longer period of time to show any response in production performance and nutrient digestibility. We concluded that supplementing xylanase to cows fed corn or sorghum silage-based diets did not improve fiber digestion. But for feeding hulled or hulless barley grains to lactating dairy cows, increased NDF concentration in diets is not necessary and hulless barley is good as corn grain for feeding lactating dairy cows as the grain source. / Ph. D. / The overall objective of this study was to evaluate the effects of feeding hulless barley and supplementing a xylanase enzyme on production performance and nutrient utilization of lactating dairy cows. Barley starch is fermented faster than corn starch and can possibly reduce ruminal pH. Reduced ruminal pH can compromise cow production performance and cause some health problems. In study 1, we evaluated production performance, milk fatty acid composition, and nutrient digestibility in high-producing dairy cows consuming diets containing corn or hulless barley as the grain source. We hypothesized that a plausible reduction in production performance and milk fat percentage would be reduced by feeding hulless barley as the grain source in the diet. According to our results, the inclusion of hulless barley grain as the energy source in diets for lactating dairy cows resulted in similar production performance and nutrient utilization as corn grain. We concluded that hulless barley is as good as corn grain as an energy source and increasing fiber concentration in hulless barley-based diet is not necessary. In study 2, we evaluated production performance, nutrient digestibility, and milk fatty acid composition of high-producing dairy cows consuming diets containing hulled or hulless barley as the grain source. We hypothesized that rumen function is altered when cows are fed low-forage diets containing barley grains, and this altered rumen function would be reflected in lower production performance and a reduction of milk fat percentage. Contrary to our expectations, we did not observe any differences in cow production performance among all treatments. We concluded that both hulled or hulless barley grains are good energy sources for sustaining high milk production and there is no need to increase fiber concentration in diet when using barley grain as the grain source. In study 3, we evaluated the effects of supplementing a xylanase enzyme on production performance and nutrient digestibility of lactating dairy cows fed diets containing corn or sorghum silage as the forage source. We hypothesized that supplementing a xylanase enzyme product in diets containing corn or sorghum silage increases fiber digestibility, and production performance of lactating dairy cows would also be improved due to enhanced fiber digestion. Supplementation of xylanase for 19 d did not affect cow production performance and nutrient digestion. The effects of supplementation xylanase may require a longer period time to detect. We concluded that supplementing xylanase to cows fed corn or sorghum silage-based diets did not improve fiber digestion. For feeding hulled or hulless barley grains to lactating dairy cows, increased fiber concentration in diets is not necessary and hulless barley is good as corn grain for feeding lactating dairy cows as the grain source.

Xylanase was purified from the crude culture of Aspergillus oryzae DSM1863 by sephadex G200 and DEAE – cellulose ion exchange chromatography. The molecular mass of the purified xylanase determined by SDS–PAGE was 21 kDa with a specific activity of 6768 U/mg towards 1% (w/v) of birch wood xylan. The optimum temperature was observed at 60°C. The enzyme was thermostable in the temperature range of 37-50°C with a high residual activity of 62-74% (650.6- 775.9 U/mg protein). / Enzyme xylanase được tinh sạch từ dịch lên men của chủng Aspergillus oryzae DSM1863 sau khi qua cột sắc ký lọc gel sephadex G200 và sắc ký trao đổi ion DEAE – cellulose. Khối lượng phân tử của enzyme xylanase tinh sạch được xác định bằng điên di đồ SDS- PAGE. Xylanase tinh sạch có kích thước là 21 kDa với hoạt tính đặc hiệu đạt 6768 U/mg sau khi được xác định với nồng độ cơ chất là 1% birch wood xylan. Nhiệt độ tối ưu để enzyme hoạt động mạnh nhất là 60°C. Enzyme xylanase khá bền nhiệt. Hoạt tính của enzyme vẫn còn duy trì 62-74% (hoạt tính đặc hiệu đạt 650.6-775.9 U/mg protein) sau khi 8 giờ ủ ở 37-50°C.

Parmi les ressources d'origines agricole et forestière utilisables aujourd'hui en tant que biomasse à destination énergétique, le miscanthus apparait comme l'une des espèces de graminées les plus prometteuses pour la production de bioéthanol de seconde génération grâce à son haut potentiel en biomasse. Ce procédé dit "2G" convertit la cellulose contenue dans ces biomasses lignocellulosiques en bioéthanol et ce via un procédé intégrant prétraitement physico-chimique, hydrolyse enzymatique et fermentation. Le principal objectif de ce projet de thèse visait à étudier l'impact de l'hétérogénéité tissulaire et structurale du miscanthus sur sa saccharification et s'est décliné en différents volets liés à l'étude de l'efficacité des prétraitements et à l'analyse des performances de différents cocktails enzymatiques de Trichoderma reesei. L'hydrolyse enzymatique est essentiellement limitée par la structure et la porosité des complexes pariétaux qui réduisent l'accessibilité de la cellulose aux cellulases. En plus des constituants hémicelluloses et lignines qui recouvrent la cellulose, les parois cellulaires du miscanthus sont riches en acides hydroxycinnamiques (pCA et FA) qui jouent un rôle important dans la cohésion du réseau pariétal complexe. L'application de prétraitements acide et alcalin sur le miscanthus a ainsi révélé une différence de réactivité en fonction des types cellulaires. Les parois secondaires du sclérenchyme sont plus facilement dégradées par les cellulases fongiques après prétraitement acide. L'étude de la distribution des composés phénoliques au niveau cellulaire par micro spectrophotométrie UV a rapporté une nette diminution de l'absorbance UV dans tous les tissus après chaque prétraitement. Ceci n'expliquant pas totalement les différences de réactivité observées, d'autres facteurs physicochimiques seraient donc impliqués. Une approche visant à évaluer la progression des cellulases au sein des parois par immunocytochimie a également été initiée mais elle s'est heurtée à des problématiques techniques liées à la nature des tissus et aux anticorps employés. Les performances en terme de conversion de la cellulose ont été évaluées avec des cocktails enzymatiques de T. reesei comprenant des activités (hemi-)cellulolytiques variables. Une meilleure efficacité du prétraitement par explosion à la vapeur a ainsi pu être montrée par réduction de la quantité d'enzymes mises en œuvre. Comme c'est le cas pour d'autres graminées, ces travaux ont permis de confirmer le rôle crucial de l'enzyme β-glucosidase, permettant de limiter l'inhibition par le cellobiose et améliorant la cinétique initiale de saccharification. L'amélioration du rendement d'hydrolyse par l'utilisation d'un sécrétome comprenant une bonne activité hémicellulolytique a pu être ensuite démontrée. L'utilisation de cocktails enzymatiques reconstitués à partir d'enzymes pures a enfin permis de définir un mélange "optimal" composé des quatre principales cellulases de T. reesei (CBH1, CBH2, EG1 et EG2) associées à une hémicellulase (XYN1). / Among agricultural and forest resources, the grass specie miscanthus has emerged as one of the most promising feedstock candidates for 2G-biofuel production due to its high biomass yield. The biofuels 2G-production process is based on cellulose conversion into bioethanol via physicochemical pretreatment, enzymatic hydrolysis and fermentation. The main objective of this Ph.D. project was to evaluate the effect of tissue and structure heterogeneity of miscanthus on its saccharification by evaluating pretreatment efficiency and analyzing the performance of different Trichoderma reesei cellulolytic cocktails.Enzymatic hydrolysis is mainly hindered by cell wall structure and porosity which limit cellulose accessibility to cellulase. In addition to hemicelluloses and lignin polymers, miscanthus cell walls, contain high amounts of hydroxycinnamic acids (pCA and FA) that play a significant role in cross-linking polymers into cohesive network. Applying acid and alkali pretreatments on miscanthus revealed a distinctive reactivity depending on cell types. Secondary cell walls of sclerenchyma appeared more digested by fungal cellulases after acid pretreatment. Addressing phenolics distribution (lignin and hydroxycinnamic acids) at cell level by UV micro spectrophotometry highlighted a significant decrease in UV absorbance after both pretreatments irrespective to cell type indicating that other physicochemical and structural features are involved in distinct cell wall reactivity. We have also attempted to evaluate cellulase progression into miscanthus cell walls by immunocytochemistry but we have had many technical problems due to the nature of miscanthus tissues and used antibodies. Cellulose conversion ability was then evaluated using enzymatic cocktails of T. reesei which vary in their (hemi-)cellulolytic activities. Higher efficiency of the steam explosion pretreatment was demonstrated by reducing enzymes loading. As reported previously on other grasses, β-glucosidase plays a crucial role by limiting the inhibiting effect of cellobiose and improving the initial saccharification step. We furthermore showed that the use of hemicellulases-improved cocktails allowed significant increase in saccharification yields. We finally identified an optimal reconstituted enzyme mixture composed of four major cellulases of T. reesei (CBH1, CBH2, EG1 and EG2) and the hemicellulase XYN-1.

A maceração do milho é uma das etapas mais importantes para seu processamento através da moagem por via úmida. Consiste em uma série de processos que envolvem fermentação e hidrólise química de forma a permitir a separação adequada de seus componentes. Trata-se de uma etapa demorada que requer alto investimento em tanques e utilidades. No presente trabalho buscou-se avaliar o uso de enzimas para auxiliar na redução do tempo de maceração de milho dentado, em escala laboratorial, produzido na região do triângulo mineiro. Para tal, os grãos foram submetidos a diferentes tratamentos com as enzimas xilanase e protease. A ação enzimática foi comparada a padrões de maceração convencionais. Os resultados obtidos mostraram que o uso de enzima permite diminuir o tempo de maceração em torno de 18 horas assim como reduzir a quantidade de SO2 adicionada ao processo de maceração. / Corn steeping is one of the most important stage in the corn wet milling process. It consists of several processes involving fermentation and chemical hidrolysis to enable separation of different fractions of the grain. This process is time and capital investment intense. In the present work it was studied the use of enzymes to reduce the steeping time for dent corn, produced in the southeast of Brazil, in laboratory scale. The evaluated enzymes were xilanase and protease. The enzyme performance was compared with the regular steeping process. The final result has shown that the use of enzymes enabled to reduce steeping time in 18 hours as weel as the amount of added SO2 in the steeping process.

Os ingredientes de origem vegetal que compõem as dietas de frangos de corte possuem em suas composições uma fração indigestível, os polissacarídeos não amiláceos (PNAs). Estas macromoléculas poliméricas de açúcares simples (monossacarídeos) são resistentes à hidrólise no trato gastrointestinal dos animais monogástricos, caracterizando-se como fatores antinutricionais. O objetivo deste trabalho foi avaliar o desempenho zootécnico e rendimento de carcaça de frangos de corte criados de 1 a 42 dias de idade, alimentados com dietas à base de milho ou trigo, com ou sem inclusão de xilanase e valorização da matriz energética. No experimento foram utilizados 1.152 frangos de corte machos, da linhagem Cobb 500. Os animais foram distribuídos em blocos casualizados, com os tratamentos em esquema fatorial 2 x 2 x 2 (duas rações : uma com milho e outra com trigo, 2 níveis de xilanase: 0 e 100 g/ton e 2 níveis de energia: de acordo com as recomendações para cada fase e reduzido em 60 kcal/kg em relação às recomendações), totalizando oito tratamentos com 12 repetições de 12 aves por unidade experimental. As dietas experimentais, com milho ou trigo, foram formuladas para atender as exigências nutricionais das aves segundo Rostagno et al. (2011). Os dados de rendimento de carcaça e desempenho de crescimento foram analisados pelo software SAS 9.3, com análise de variância, e as médias comparadas pelo teste de Tukey (P 0,05). Os resultados indicaram melhorias nos parâmetros de desempenho com a adição de enzima em dietas com base de trigo, e melhor conversão alimentar aos 42 dias de idade com o uso da enzima nas dietas com nível de energia padrão / Ingredients of vegetal sources, which compose the diets of broilers, have in its composition an indigestible fraction known as the non-starch polysaccharides (NSP). These polimeric macromolecules of simple sugars (monosaccharides) are resistant to hydrolisis in the gastrointestinal tract of monogastric animals, and are characterized as anti-nutritional factors. The objective of this work was to evaluate the performance and the carcass yield of broilers raised from 1 to 42 days of age, fed with diets based in corn or wheat, with or without inclusion of xylanase and valorization of the energetic matrix. In the experiment it were used 1.152 male broilers, from the Cobb 500 lineage. The animals were distributed in randomized blocks, with treatments in a factorial 2 x 2 x 2 (two feeds: one with corn and the other with wheat, 2 levels of xylanase: 0 and 100 g/ton, and 2 levels of energy: according to recommendations for each phase, and reduced in 60 kcal/kg in relation to the recommendations), completing eight treatments with twelve replications, and twelve birds in each experimental unit. The experimental diets, with corn or wheat, were formulated to supply the nutritional requirements of the birds according to Rostagno et al. (2011). Carcass yield and growth performance data were analyzed by SAS 9.3 software, with analysis of variance, and the means compared by the Tukey test (P 0.05). Results presented improvements in parameters of performance with inclusion of the enzyme in diets based in wheat, and best feed conversion rate for the period of 1 to 42 days of age with the use of enzyme on standard level energy of diet