Nas células do ceco anterior de A.flavolineata, a secreção de enzimas digestivas parece ser mediada por vesículas de secreção e é influenciada pelo tempo decorrido após a refeição. Fracionamentos subcelulares das células do ceco anterior realizados 3h após a refeição indicaram que a amilase, maltase, pNΦβglu hidrolase e aminopeptidase estão enriquecidas nas frações que contém os grânulos de secreção. γ glutamil transferase, aminopeptidase e dipeptidase apresentaram uma forma solúvel e outra ligada a membrana. No conteúdo do intestino de A. flavolineata foi possível detectar a presença de 3 β-glucosidases: 1, uma celobiase-aril β-glucosidase termoestável; 2, uma aril β-glucosidase termoinstável ativa sobre pNΦβglu; 3, uma alquil β-glucosidase. A celobiase-aril β-glucosidase hidrolisa celobiose e aril β-glucosídeos em sítios diferentes e é mais ativa sobre celobiose e laminaribiose que sobre aril β-glucosídeos sintéticos ou naturais. Moléculas anfipáticas ativam a alquil β-glucosidase, tornando esta enzima efetivamente ativa apenas na digestão de membranas. Esta enzima hidrolisa alquil β-glucosídeos com 6 a 11 carbonos no radical alquil. A celobiase-aril P-glucosidase e a alquil β-glucosidase são provavelmente responsáveis pela digestão \"in vivo\" de β-1,4; β-1,3; β-1,3-1,4 glucanas e glucosilceramidas, respectivamente. / The secretion of digestive enzymes in the anterior caecal cells of A. flavolineata seems to be mediated by secretory vesicles and influenced by the period of time after a meal. Subcellular fractions of anterior caecal cells were obtained by differential centrifugation of homogenates prepared 3 hours after a meal. Amylase, maltase, pNΦβglu hidrolase and aminopeptidase are found with high activities in fractions that correspond to the contents of secretory vesicles. γ glutamil transferase, aminopeptidase and dipeptidase presented soluble and membrane-bound forms. In A. flavolineata midgut contents we found 3 β-glucosidases: 1, a heat-stable cellobiase-aryl β-glucosidase; 2, a heat-unstable activity against pNΦ β glu (aryl β-glucosidase); 3, a alkyl β-glucosidase. The cellobiase-aryl β-glucosidase hydrolyzes cellobiose and aryl β-glucosides at different active sites and is more active on cellobiose and laminaribiose than on aryl β-glucosides. Amphipatic molecules activate the alkyl β-glucosidase, making the enzyme very active only during membrane digestion. This enzyme hydrolyzes alkyl β-glucosides with 6 to 11 carbons in alkyl moiety. The cellobiase-aryl β-glucosidase and alkyl β-glucosidase are probably responsible for in vivo digestion of β-1,4; β-1,3; β-1,3-1,4 glucans and glucosylceramides, respectively.

La levure Saccharomyces cerevisiae est capable d’utiliser une grande variété de sucres comme source de carbone et d’énergie. La plupart des enzymes impliquées dans l’utilisation de ces sucres sont codées par des gènes issus de familles multigéniques. C’est le cas de la famille IMA identifiée comme impliquée dans l’utilisation de l’isomaltose. Cette famille comprend cinq gènes qui codent pour quatre isomaltases partageant une forte identité de séquence (de 65% à 100 %). Dans ce travail , la diversitéfonctionnelle de la famille IMA a été étudiée, en caractérisant de façon exhaustive in vitro leurs propriétés biochimiques et enzymologiques. Ima1p et Ima2p possèdent des propriétés biochimiques identiques (pH, température, et thermostabilité) mais Ima3p se distingue par rapport à ces deux protéines bien que n’ayant que trois acides de différence avec Ima2p (thermostabilité plus faible). Ima5p quant à elle, est la protéine la plus dissemblable (température optimale plus faible et demi-vie basse dès 37°C). Les quatre isomaltases sont cependant très sensibles au Tris et aux ions Fe3+. Les quatre isoenzymes présentent une préférence pour les disaccharides liés en α-1,6 (isomaltose et palatinose), avec une cinétique de type Michaëlis-Menten et une inhibition par le substrat à une concentration élevée. Les isomaltases Imap sont cependant aussi capables d'hydrolyser les disaccharides α-1,2, α-1,3 et α-1,5 ainsi que les trisaccharides portant une liaison α-1,6, ce qui met en évidence leur ambiguïté de substrat .Nos résultats ont toutefois montré de nombreuses singularités dans cette famille de protéines. Alors que Ima1p et Ima2p présentent des propriétés très semblables, l’activité catalytique de Ima3p est globalement très faible malgré sa forte ressemblance avec Ima2p. Le variant Ima3p_R279Q retrouve des niveaux d'activité proches de ceux d’Ima2p, tandis que la substitution d’une leucine par une proline à la position 240 a permis d’augmenter de manière significative la stabilité d’Ima3p confirmant le rôle des prolines dans la thermostabilité des protéines. L’hydrolyse de l’isomaltose par Ima5p réfute lesconclusions précédemment publiées sur l'exigence d'acides aminés spécifiques pour déterminer la spécificité de α-1,6 puisque le variant IMA5-MQH ne permet pas de restaurer une activité semblable à Ima1p malgré la présence des trois résidus MQH. Nous avons également trouvé qu’Ima5p est inhibé par le maltose suivant une inhibition mixte tandis qu’Ima1p est inhibée de façon compétitive à faible concentration et de manière incompétitive à forte concentration en isomaltose / Most enzymatic systems for sugar uptake and assimilation rely on multigene families in theyeast Saccharomyces cerevisiae. The IMA / MAL family has been used as a model system to study themolecular mechanisms that govern evolution of duplicated genes. The five IMA multigene familymembers encode four isomaltases sharing high sequence identity from 65% to 99%, of which IMA3and IMA4 are 100% identical to encode the same isomaltase. In this work, the functional diversity ofIMA family was further explored, with exhaustive in-vitro characterization of their biochemical andenzymological properties.Ima1p and Ima2p were similar to biochemical properties; Ima3p showed some differences fromthe two proteins; amongst them, Ima5p was the most distant protein. The four isomaltases were highlysensitive to Tris and Fe3+, but were unaffected by the addition or the removal of Ca2+ despiteconservation of the calcium binding site. Besides, four isoenzymes exhibited a preference for the α-(1,6)disaccharides isomaltose and palatinose, with Michaelis-Menten kinetics and inhibition at highsubstrates concentration. They were also able to hydrolyse trisaccharides bearing an α-(1,6) linkage,but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrateambiguity. While Ima1p and Ima2p presented almost identical characteristics, the results neverthelessshowed many singularities within this protein family. In particular, Ima3p presented lower activitiesthan Ima2p despite only 3 different amino acids between these two isoforms. The Ima3p_R279Qvariant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240significantly increased the stability of Ima3p and supported the role of prolines inthermostability.Ima5p presented the lower optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specificamino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibitionby maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitiveat very low isomaltose concentrations and uncompetitive as the substrate concentration increased.The presented Ph.D’s work provided preliminary insights into determining structural factorswithin this family, exemplifying for example the role of proline residues for thermosability. Moreover,it was illustrated that a gene family encoding proteins with strong sequence similarities can lead toenzyme with notable differences in biochemical and enzymological properties.

Glucosidases são enzimas que catalisam a hidrólise de ligações glicosídicas liberando unidades monossacarídicas de um terminal não redutor de um oligossacarídeo ou glicoconjugado. Iminoaçúcares são alcalóides piperidínicos polihidroxilados isolados de plantas (gênero Morus) e microrganismos (Bacillus), como nojirimicina (NJ) (1) e 1-desoxinojirimicina (DNJ) (2), os quais são descritos como inibidores de glucosidase. O potencial uso destes inibidores no tratamento de infecções virais, crescimento tumoral, metástases, diabetes, doença de Gaucher e osteoartrite tem motivado a comunidade científica na busca por novos derivados iminoaçúcares. Desse modo, a síntese de pseudodissacarídeos, contendo ambos resíduos de iminoaçúcar e glicopiranose, constitui uma estratégia interessante de obtenção desses derivados, apesar dos desafios envolvidos na geração da ligação entre estes dois açúcares. Por esta razão, foi utilizada a estratégia de click chemistry como uma ferramenta para introduzir uma ponte de grupo 1,2,3-triazol entre os açúcares a partir do acoplamento de azido-glicosídeo com N-propargil-iminoaçúcar. Desta forma, a síntese do iminoaçúcar N-propargílico (73), com função acetileno terminal, foi realizada em cinco etapas e foi usado na reação de cicloadição 1,3- dipolar com três derivados glicosídicos contendo grupo azido nas posições anomérica (C-1), C-3 ou C-6. A partir desta reação CuAAC (Copper(I)-catalyzed Azide-Alkyne Cycloaddition), três novos pseudo-dissacarídeos (77, 81 e 85) foram sintetizados em rendimentos moderados e foram, então, avaliados em ensaios de - D-glucosidase isolada de Sacharomyces cerevisiae. Nestes testes preliminares, o composto 77 foi o mais ativo, o qual foi capaz de inibir a atividade da enzima em 40% a 1mM. Esses resultados encorajam a realização de novos experimentos, principalmente, a determinação de Ki e avaliação da atividade relativa à replicação do vírus HIV. Portanto, a obtenção destes pseudodissacarídeos trouxe uma contribuição importante no que diz respeito à química de carboidratos e também ao tratamento das doenças citadas. / Glucosidases are enzymes that catalyze the hydrolysis of glycosidic bonds releasing monosaccharide units from a non-reducing end of an oligosaccharide or glycoconjugate. Iminosugars are polihydroxilate piperidinic alkaloids isolated from plants (Morus alba) and microorganisms (Bacillus), such as nojirimicin (NJ) (1) and 1- deoxynojirimicin (2), which are described as glucosidase inhibitors. The potential use of these inhibitors in the treatment of viral infection, tumoral growing, metastasis, diabetes, Gaucher´s disease and osteoarthritis has stimulated the scientific community on the search for novel iminosugar derivatives. Thereby, the synthesis of pseudodisaccharides, having both iminosugar and glycopyranose residues, represents an interesting strategy to obtain these derivatives, despite the challenges involved in generating the link between these two sugars. For this reason, we have used click chemistry as a tool to introduce a 1,2,3-triazole bridge between the sugars from the coupling of azide-glycosides with N-propargyl-iminosugar. Thus, the synthesis of N-propargyl-iminosugar (73), containing the terminal acetylene function, was performed in five steps, and was used in the 1,3-dipolar cycloaddition reaction with three glycosidic derivatives containing the azide group at anomeric (C-1), C-3 or C-6 positions. By applying this CuAAC (Copper(I)-catalyzed Azide-Alkyne Cycloaddition), three novel pseudo-disaccharides (77, 81 and 85) were synthesized in moderate yields and then, evaluated in -D-glucosidase assays isolated from Sacharomyces cerevisiae. In these preliminary test, compound 77 was the most active from the series, which was able to inhibit 40% of the enzyme activity at 1 mM. These results encourage us to perform new experiments, notably the determination of Ki and evaluation towards HIV replication. Thus, a contribution regarding carbohydrate chemistry and treatment of the supracited diseases was achieved by the synthesis of these pseudodisaccharides.

Glucosidases são enzimas que catalisam a hidrólise de ligações glicosídicas liberando unidades monossacarídicas de um terminal não redutor de um oligossacarídeo ou glicoconjugado. Iminoaçúcares são alcalóides piperidínicos polihidroxilados isolados de plantas (gênero Morus) e microrganismos (Bacillus), como nojirimicina (NJ) (1) e 1-desoxinojirimicina (DNJ) (2), os quais são descritos como inibidores de glucosidase. O potencial uso destes inibidores no tratamento de infecções virais, crescimento tumoral, metástases, diabetes, doença de Gaucher e osteoartrite tem motivado a comunidade científica na busca por novos derivados iminoaçúcares. Desse modo, a síntese de pseudodissacarídeos, contendo ambos resíduos de iminoaçúcar e glicopiranose, constitui uma estratégia interessante de obtenção desses derivados, apesar dos desafios envolvidos na geração da ligação entre estes dois açúcares. Por esta razão, foi utilizada a estratégia de click chemistry como uma ferramenta para introduzir uma ponte de grupo 1,2,3-triazol entre os açúcares a partir do acoplamento de azido-glicosídeo com N-propargil-iminoaçúcar. Desta forma, a síntese do iminoaçúcar N-propargílico (73), com função acetileno terminal, foi realizada em cinco etapas e foi usado na reação de cicloadição 1,3- dipolar com três derivados glicosídicos contendo grupo azido nas posições anomérica (C-1), C-3 ou C-6. A partir desta reação CuAAC (Copper(I)-catalyzed Azide-Alkyne Cycloaddition), três novos pseudo-dissacarídeos (77, 81 e 85) foram sintetizados em rendimentos moderados e foram, então, avaliados em ensaios de - D-glucosidase isolada de Sacharomyces cerevisiae. Nestes testes preliminares, o composto 77 foi o mais ativo, o qual foi capaz de inibir a atividade da enzima em 40% a 1mM. Esses resultados encorajam a realização de novos experimentos, principalmente, a determinação de Ki e avaliação da atividade relativa à replicação do vírus HIV. Portanto, a obtenção destes pseudodissacarídeos trouxe uma contribuição importante no que diz respeito à química de carboidratos e também ao tratamento das doenças citadas. / Glucosidases are enzymes that catalyze the hydrolysis of glycosidic bonds releasing monosaccharide units from a non-reducing end of an oligosaccharide or glycoconjugate. Iminosugars are polihydroxilate piperidinic alkaloids isolated from plants (Morus alba) and microorganisms (Bacillus), such as nojirimicin (NJ) (1) and 1- deoxynojirimicin (2), which are described as glucosidase inhibitors. The potential use of these inhibitors in the treatment of viral infection, tumoral growing, metastasis, diabetes, Gaucher´s disease and osteoarthritis has stimulated the scientific community on the search for novel iminosugar derivatives. Thereby, the synthesis of pseudodisaccharides, having both iminosugar and glycopyranose residues, represents an interesting strategy to obtain these derivatives, despite the challenges involved in generating the link between these two sugars. For this reason, we have used click chemistry as a tool to introduce a 1,2,3-triazole bridge between the sugars from the coupling of azide-glycosides with N-propargyl-iminosugar. Thus, the synthesis of N-propargyl-iminosugar (73), containing the terminal acetylene function, was performed in five steps, and was used in the 1,3-dipolar cycloaddition reaction with three glycosidic derivatives containing the azide group at anomeric (C-1), C-3 or C-6 positions. By applying this CuAAC (Copper(I)-catalyzed Azide-Alkyne Cycloaddition), three novel pseudo-disaccharides (77, 81 and 85) were synthesized in moderate yields and then, evaluated in -D-glucosidase assays isolated from Sacharomyces cerevisiae. In these preliminary test, compound 77 was the most active from the series, which was able to inhibit 40% of the enzyme activity at 1 mM. These results encourage us to perform new experiments, notably the determination of Ki and evaluation towards HIV replication. Thus, a contribution regarding carbohydrate chemistry and treatment of the supracited diseases was achieved by the synthesis of these pseudodisaccharides.

In order to improve the production of acarbose, the fermentation medium of acarbose-producing strain Actinoplanes sp. KCTC 9161 – L14 mutant was optimized in this internship. Fractional factorial design was employ to investigate the influences of glucose, maltose and corn power on acarbose production (by a-glucosidase inhibitory ability). Two significant factors: glucose and maltose have significant and positive effects on acarbose amount. In addition, a model was obtained from the regression results of fractional factorial experiment. Other success, we demonstrated that chromatography by active charcoal column can used to purify acarbose from fermentation broth. Acarbose amount in purification solution was 191.5 g/L and an acarbose - purification process was inducted. / Nhằm mục đích nâng cao khả năng sinh tổng hợp hoạt chất acarbose từ chủng đột biến Actinoplanes sp. KCTC 9161-L14, môi trường lên men của chủng dùng để sản xuất acarbose đã được tối ưu hóa. Một phần mềm thiết kế đã được thiết lập để khảo sát ảnh hưởng của glucose, maltose và bột ngô đến khả năng sản xuất acarbose (thông qua hoạt tính ức chế a-glucosidase). Kết quả đã cho thấy, hai yếu tố glucose và maltose có ý nghĩa quan trọng và ảnh hưởng trực tiếp đến khả năng sinh tổng hợp acarbose. Một phương trình đã được hình thành từ kết quả tối ưu. Bên cạnh đó, chúng tôi đã chứng minh được cột sắc ký sử dụng than hoạt tính có thể tinh sạch acarbose từ dịch lên men. Hàm lượng acarbose trong dung dịch tinh sạch đạt 191,5 g/l và một quy trình tinh sạch acarbose được đề xuất.

In order to improve the production of acarbose, the fermentation medium of acarbose-producing strain Actinoplanes sp. KCTC 9161 – L14 mutant was optimized in this internship. Fractional factorial design was employ to investigate the influences of glucose, maltose and corn power on acarbose production (by a-glucosidase inhibitory ability). Two significant factors: glucose and maltose have significant and positive effects on acarbose amount. In addition, a model was obtained from the regression results of fractional factorial experiment. Other success, we demonstrated that chromatography by active charcoal column can used to purify acarbose from fermentation broth. Acarbose amount in purification solution was 191.5 g/L and an acarbose - purification process was inducted. / Nhằm mục đích nâng cao khả năng sinh tổng hợp hoạt chất acarbose từ chủng đột biến Actinoplanes sp. KCTC 9161-L14, môi trường lên men của chủng dùng để sản xuất acarbose đã được tối ưu hóa. Một phần mềm thiết kế đã được thiết lập để khảo sát ảnh hưởng của glucose, maltose và bột ngô đến khả năng sản xuất acarbose (thông qua hoạt tính ức chế a-glucosidase). Kết quả đã cho thấy, hai yếu tố glucose và maltose có ý nghĩa quan trọng và ảnh hưởng trực tiếp đến khả năng sinh tổng hợp acarbose. Một phương trình đã được hình thành từ kết quả tối ưu. Bên cạnh đó, chúng tôi đã chứng minh được cột sắc ký sử dụng than hoạt tính có thể tinh sạch acarbose từ dịch lên men. Hàm lượng acarbose trong dung dịch tinh sạch đạt 191,5 g/l và một quy trình tinh sạch acarbose được đề xuất.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Humanity is currently dependant on fossil fuels as an energy source. Increasing economic development and industrialization is, however, raising the demand for this unsustainable energy source. This increased pressure on dwindling reserves and growing concern over detrimental environmental effects associated with the use of these fuels have sparked great interest in the development of alternative sources. Bioethanol has surfaced as a good alternative to fossil fuels, as it can be produced from cheap, abundant, renewable, non-food sources. Bioethanol is also carbonneutral, i.e. utilisation thereof leaves the net level of carbon dioxide in the atmosphere unperturbed. Lignocellulose, more specifically its cellulose fraction, has been identified as a possible feedstock for the production of bioethanol. The use of lignocellulose as feedstock will allow for a more sustainable supply and much needed energy security. Lignocellulosic feedstocks can be divided into two main categories, i.e. wastes from processes other than fuel production and crops grown specifically for fuel production. Cereal crops such as triticale have been identified as good industrial crops for the production of energy. Triticale’s higher biomass yield, moderate water and nutrient requirements, steadily increasing area of cultivation and main use as an animal feed and not a human food source, makes it attractive as feedstock for the production of bioethanol. The combined activity of endoglucanases, exoglucanases and β-glucosidases is needed to hydrolyse crystalline cellulose to fermentable sugars. The high cost of these enzymes is, however, the most significant barrier to the economical production of bioethanol from cellulosic biomass. A promising strategy for a reduction in costs is the production of these cellulolytic enzymes, hydrolysis of biomass and fermentation of the resulting sugars to bioethanol in a single process step via a cellulolytic microorganism. The development of such a consolidated bioprocessing (CBP) organism can be achieved by the introduction of cellulolytic activity into a noncellulolytic microorganism that is able to ferment glucose to ethanol. Saccharomyces cerevisiae is a good host candidate for CBP as this yeast’s high tolerance towards ethanol and its use in industrial applications has been established. The enzymatic activities of endoglucanases and exoglucanases are, however, inhibited by the build-up of cellobiose during the hydrolysis of cellulose. This effect may be alleviated with the introduction of a better functioning β-glucosidase into the system. β-Glucosidases hydrolyse cellobiose to glucose, alleviating the inhibition on the enzymatic activities of endoglucanases and exoglucanases. Despite advances in enzyme production systems and engineering enzymes currently in use for higher stability and activity, there is still a demand to expand the current collection of enzymes. Bioprospecting for novel cellulolytic enzymes focuses on specific environment, with high turnover rates of cellulosic material or extreme conditions, such as the composting process. These enzymes are becoming more attractive compared to their mesophillic counterparts due to their potential industrial applications and the fact that they represent the lower natural limits of protein stability. / AFRIKAANSE OPSOMMING: Die mensdom is hoofsaaklik van fossielbrandstowwe as 'n energiebron afhanklik. Toenemende ekonomiese ontwikkeling en industrialisasie verhoog egter die aanvraag na hierdie onvolhoubare energiebron. Druk op kwynende reserwes en groeiende kommer oor die nadelige gevolge vir die omgewing wat met die gebruik van hierdie brandstowwe gepaard gaan, het tot groot belangstelling in die ontwikkeling van alternatiewe bronne gelei. Bio-etanol is 'n goeie alternatief vir fossielbrandstowwe, want dit kan van goedkoop, vollop, hernubare nievoedselbronne geproduseer word. Bio-etanol is ook koolstof-neutraal; die gebruik daarvan laat die netto vlak van koolstofdioksied in die atmosfeer onverstoord. Lignosellulose, en meer spesifiek die sellulose fraksie, is as moontlike grondstof vir die vervaardiging van bio-etanol geïdentifiseer. Die gebruik van lignosellulose as grondstof sal meer volhoubare voorsiening en broodnodige energie-sekuriteit verseker. Sellulose grondstowwe kan in twee hoof kategorieë verdeel word, nl. Newe produkteafval van prosesse anders as brandstofproduksie en gewasse wat spesifiek vir brandstofproduksie gekweek word. Graangewasse soos korog is geïdentifiseer as 'n goeie industriële gewas vir die produksie van energie. Korog se hoër biomassa opbrengs, matige water en voedingstofvereistes, groeiende bewerkingsgebied en die gebruik as 'n veevoergewas eerder as 'n menslike voedselbron, maak dit aantreklik as 'n grondstof vir die vervaardiging van bio-etanol. Die gesamentlike aktiwiteit van endoglukanases, eksoglukanases en β-glukosidases is nodig om kristallyne sellulose tot fermenteerbare suikers te hidroliseer. Die hoë koste van hierdie ensieme is egter die grootste hindernis vir die ekonomiese produksie van bio-etanol vanaf sellulosiese biomassa. 'n Belowende koste verminderingstrategie is die produksie van hierdie sellulolitiese ensieme, die hidrolise van biomassa, en die fermentasie van die suikers na bio-etanol in 'n enkelstap-proses via 'n sellulolitiese mikro-organisme. Die ontwikkeling van so 'n gekonsolideerde bioprosesserings (CBP) organisme kan deur die uitdrukking van sellulolitiese aktiwiteite in 'n nie-sellulolitiese mikro-organisme wat wel in staat is om glukose na etanol om te fermenteer, gerealiseer word. Saccharomyces cerevisiae is 'n goeie kandidaat gasheer vir CBP, omdat hierdie gis ‘n hoë verdraagsaamheid teenoor etanol toon en sy gebruik in industriële toepassings gevestig is. Die ensiematiese aktiwiteite van endoglukanases en eksoglukanases word egter deur die ophoop van sellobiose gedurende die hidrolise van sellulose geïnhibeer. Hierdie effek kan met die byvoeging van meer effektiewe β-glukosidases verlig word. β-Glukosidases hidroliseer sellobiose na glukose en verlig dus die inhibisie op die endoglukanase en eksoglukanase ensiematiese aktiwiteite. Ten spyte van vooruitgang in ensiemproduksie stelsels en ensiemmodifiserings strategieë wat tans vir hoër stabiliteit en aktiwiteit in gebruik is, bestaan daar steeds 'n behoefte om die bestaande versameling van ensieme uit te brei. Bioprospektering vir nuwe sellulolitiese ensieme fokus op spesifieke omgewings, met hoë omsetkoerse van sellulose materiaal of omgewings met uiterste toestande, soos die komposterings-proses. Hierdie ensieme is besig om meer aantreklik in vergelyking met hul mesofieliese eweknieë te raak as gevolg van hul potensiele industriële toepassings en die feit dat hulle die laer natuurlike grense van proteïen-stabiliteit verteenwoordig. / Stellenbosch University and the Technology Innovation Agency for financial support

?-glucosidases são enzimas que catalisam a hidrólise de ligações glucosídicas ?-1,4, ?-1,3 e ?-1,6 a partir da extremidade não redutora de oligossacarídeos de cadeias pequenas, alquil e aril ?-D-glucosídeos e dissacarídeos. Além de serem enzimas chave do complexo celulolítico, apresentam funções importantes como o melhoramento de aromas de vinhos e a hidrólise de antocianinas. Malbranchea pulchella usualmente é encontrado em fragmentos vegetais em decomposição ou material rico em celulose, podendo ser considerado promissor à produção de enzimas de interesse biotecnológico. Neste contexto, o objetivo desse projeto foi a caracterização funcional de uma ?-glucosidase de M. pulchella e sua aplicação na hidrólise de resíduos agroindustriais e de antocianinas. Uma BGL da família GH3 foi purificada com um fator de purificação 6,32 e recuperação de aproximadamente 35 %. A sua massa molecular aproximado foi de 100 kDa, e o Km, Vmáx e Kcat foram calculadas em 0,33 mM, 13,67 U/mg, 26,5 s-1 respectivamente. O dicroísmo circular revelou uma estrutura composta por aproximadamente 25% de ?-hélices e 20% de ?-folhas. A BGL apresentou pH e temperatura ótimos igual a 6,0 e 50 °C; e foi estável a 40 °C e apresentou boa estabilidade nos pH 5,0 a 8,0, por 24 horas. Nenhum dos sais de íons metálicos ativou a enzima e apenas o HgCl2 inibiu a atividade em 90%. A enzima não apresentou inibição em presença de glucose (0,1-1M) por até 24 horas. Além disso, a GH3 mostrou-se glicosilada e a proporção de açúcar corresponde a 15% massa da enzima. O efeito da celobiose (C) e do bagaço de cana-de-açúcar in natura (BCAN) na produção das BGLs foram avaliados em um DCCR, que indicou um modelo reduzido com influência das duas variáveis. A melhor condição de cultivo para a produção de BGLs foi 0,6% de C (p/v) e 4% de BCAN (p/v). Por meio de um planejamento de mistura, os resíduos BCAN, a casca de soja moída (CS) e o bagaço de cevada (BCev), foram avaliados quanto ao potencial de hidrólise a partir das enzimas presentes no extrato enzimático, resultando no maior potencial de hidrólise sobre o BCev, com a produção de aproximadamente 2 mg/mL de açúcares redutores em 48 horas. As BGLs presentes no extrato enzimático otimizado foram imobilizadas em suporte MANAE-agarose, Concanavalina A-Sepharose e BrCN-Sepharose. Os derivados BGL-MANAE e BGL-ConA foram ativados aproximadamente 10 e 3 vezes, respectivamente. BGL-MANAE e BGL-ConA foram mais estáveis que o controle BGLBrCN em todos os pH testados em 24 horas e, além disso, BGL-ConA permaneceu com 100% de sua atividade em temperaturas de 40 °C, 50 °C e 60 °C, já o BGL-MANAE mostrou-se estável a 40 °C permanecendo com 83% de sua atividade, ambos em 24 horas. BGL-MANAE e BGL-ConA apresentaram menor efeito inibitório em presença de diferentes concentrações de glucose e etanol quando comparados ao BGL-BrCN, e esses resultados indicaram que a imobilização de alguma forma colaborou para uma maior estabilidade ao pH e à temperatura, bem como ao aumento da tolerância por glucose e etanol. Os derivados puderam ser reutilizados por até 20 vezes e quando avaliados quanto à capacidade de clarificar vinhos e sucos de uva (hidrólise de antocianinas), BGL-MANAE clarificou 52% o vinho, 71% o vinho diluído, 77% o suco de uva e 56% o suco de uva diluído, e BGL-ConA em contrapartida clarificou 41% o vinho, 46% o vinho diluído, 63% o suco de uva e 23% o suco de uva diluído. BGL-MANAE foi mais eficiente que BGL-ConA na clarificação de vinhos e sucos de uva podendo ser considerado um biocatalisador promissor na hidrólise de antocianinas e, consequentemente, para a produção de vinhos brancos e rose a partir de diferentes variedades de uvas. Este trabalho pelo que sabemos, é o primeiro a usar BGLs imobilizadas com aplicação na clarificação de sucos de uva e vinhos, podendo ser considerado um trabalho inovador, e de grande importância para a indústria de alimentos e bebidas. / ?-glucosidases are enzymes that catalyze the hydrolysis of ?-1,4, ?-1,3 and ?-1,6 glucosidic linkages, from the non-reducing end of short chain oligosaccharides, alkyl and aryl ?-D-glucosides and disaccharides. Besides being complex key cellulolytic enzymes, they have important functions such as the improvement of wine flavors and anthocyanins hydrolysis. Malbranchea pulchella is usually found in decaying plant debris or in material rich in cellulose, for this reason it can be considered promising for the production of enzymes of biotechnological interest. In this context, the aim of this project was the functional characterization of a ?-glucosidase from M. pulchella and its application in the organic residues and anthocyanins hydrolysis. A BGL GH3 family produced by M. pulchella was purified with a purification factor and recovery of about 6.32 and 35 times. Its approximate molecular mass was 100 kDa, and Km, Vmax and kcat were 0.33 mM, 13.67 U/mg, 26.5 s-1, respectively. The circular dichroism revealed a structure composed of approximately 25% of ?-helix and 20% of ?-sheets. BGL presented optimum pH and temperature at 6.0 and 50 °C; and it was stable at 40 °C. It also showed good stability at pH 5.0 to 8.0, for 24 hours. None of the metal ions salts activated the enzyme and HgCl2 inhibited the activity by 90%. The enzyme showed no inhibition in the presence of glucose (0,1-1M) for 24 hours. Furthermore, it is glycosylated and the sugar proportion correspondsto 15% of the enzyme mass. The effect of cellobiose (C) and sugarcane bagasse in natura (BCAN) in the production of BGLs were evaluated in a CCRD, which indicated a reduced model of influence of the two variables. The best culture condition for BGLs production was 0.6% of C (w/v) and 4% (w/v) of BCAN. Through a mixture design, using the BCAN, ground soybean hulls (CS) and barley bagasse (BCev) were used to evaluate the potential of hydrolysis of these residues in the presence of enzymes present in the enzymatic extract, resulting in a greater efficiency of (BCev) hydrolysis, producing approximately 2 mg/mL of reducing sugars in 48 hours. The BGLs present in the optimized enzyme extract were also used in the immobilization on ionic support MANAE-agarose and affinity support Concanavalin A-Sepharose (ConA-Sepharose). The BGL-MANAE and BGL-ConA derivatives were activated approximately 10 and 3 times, respectively. BGL-MANAE and BGL-ConA were more stable than BGL-BrCN control in all pH tested within 24 hours. In addition, BGL-ConA remained 100% of its activity at 40 °C, 50 °C and 60 °C , and BGL-MANAE was stable at 40 °C and remained 83% of its activity, both in 24 hours. BGL-MANAE and BGL-ConA showed lower inhibitory effect in the presence of different glucose and ethanol concentrations when compared to BGL-BrCN and these results indicate that the immobilization, somehow, cooperated to a greater pH and temperature stability, as well as to increased tolerance by glucose and ethanol. The derivatives could be reused up to 20 times and when they were tested for their capacity to clarify wine and grape juice (anthocyanins hydrolysis), BGL-MANAE clarified 52% wine, 71% diluted wine, 77% grape juice and 56% diluted grape juice. On the other hand, BGL-ConA clarified 41% wine, 46% diluted wine, 63% grape juice and 23% diluted grape juice. BGL-MANAE was more efficient than BGL-ConA in clarifying wines and grape juices and it may be considered a promising biocatalyst in the anthocyanins hydrolysis, and consequently in the production of white and rose wines from different varieties of grapes. This work as we know, is the first to use immobilized BGLs applied in the clarification of grape juice and wine, for this reason, it can be considered an innovative work, and of great importance to the food and beverage industry

Foram purificadas através de uma combinação de cromatografias as duas &#946;- glicosidases digestivas (Mr 47.000 e 50.000 - denominadas &#946;47 e &#946;50, respectivamente) encontradas na larva de S. frugiperda. Experimentos de competição entre substratos e modificação química mostraram que a &#946;47 possui dois sítios ativos. Um desses sítios denominado aril&$946;glicosidase apresenta um subsítio -1 que liga galactose mais eficientemente do enquanto ,que o subsítio +1 prefere pequenos grupos hidrofóbicos cíclicos. O segundo sítio, denominado celobiase, possui um subsítio -1 que prefere glicose. Já a região de ligação do aglicone apresenta 4 subsítios, que ligam glicose com afinidade decrescente à medida que afastam-se do ponto de clivagem do substrato. O cDNA que codifica a &#946;50 foi clonado e sequenciado. Alinhamentos de sequência de aminoácidos, experimentos de competição entre substratos e inibição mostraram que esta enzima possui apenas um sítio ativo. O subsítio -1, cuja especificidade é controlada por uma rede de pontes de hidrogênio, foi estudado comparando-se os parâmetros cinéticos (Kcat e KcaUKm) para a hidrólise de NP&#946;glicosídeos. A região de posicionamento do aglicone, uma fenda hidrofóbica composta de 3 subsítios, foi caracterizada utilizando-se alquil &#946;-glucosídeos e oligocelodextrinas como inibidores. O alinhamento da sequência de aminoácidos da &#946;50 com outras glicosil hidrolases sugeriu quais aminoácidos participariam da ligação do substrato e que o GlU187 (doador de prótons - pKa = 7,5) e o GIU399 (nucleófilo - pKa = 4,5) estão diretamente envolvidos na catálise. Além disso, a Arg97 e a Tyr331 participam indiretamente modulando o pKa do GIU399. r . / Two digestive &#946;-glycosidases (MW 47,000 and 50,000, named &#946;gly47 and &#946;gly50, respectively) whose are found in the S. frugiperda larvae were purified by a combination of chromatographic steps. Substrate competition experiments and chemical modification data showed that &#946;gly47 has two active sites. One of them was called aryl &#946;-glycosidase and presents a -1 subsite that prefers galactose while the +1 subsite binds small cyclic hydrophobic groups. The other active site was called cellobiase and presents 4 subsites that bind glucose residues weaker as they get far from the cleavage point. The cDNA that codes the &#946;gly50 was cloned and sequenced. Amino acid sequence alignment, substrate competition experiments and inhibitions proved that this enzyme has just one active site. The -1 subsite specificity is controlled by a hydrogen bond network as it was showed comparing the kinetic parameters (Kcat and KcatlKm) for some NP&#946;glycosides hydrolysis. The aglycone binding region, a hydrophobic cleft, was studied with alkyl &#946;-glucosides and oligocellodextrins as competitive inhibitors. Amino acid sequence alignment between the &#946;gly50 and other glycosil hydrolases showed the amino acids responsible for the substrate binding and that the GIU<SUB.187 (proton donor - pKa = 7.5) and GIU399 (nucleophile - pKa = 4.5) are directly involved in the catalysis. Beside this, Arg97 and Tyr331 participate indirectly in the catalysis, modulating the nucleophile pKa

Wan Leong Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 80-83). / Acknowledgments --- p.i / Bibliography --- p.ii / Contents --- p.iii / Abstract --- p.iv / Abbreviations --- p.v / Chapter I --- Introduction / Chapter I-1 --- General Background of Pseudo-sugar --- p.1 / Chapter I-2 --- Monocarba-sugar --- p.2 / Chapter I-3 --- Dicarba-sugar --- p.4 / Chapter I-4 --- Isolation of Valiolamine and Its Related Compounds --- p.6 / Chapter I-5 --- Previous Syntheses of Valiolamine --- p.8 / Chapter II --- Results and Discussions / Chapter II-1 --- General Strategy --- p.17 / Chapter II-2 --- "Synthesis of (lR,2R)-diol (62)" --- p.20 / Chapter II-3 --- Synthesis and Reactivity of Olefin 69 --- p.24 / Chapter II-4 --- "Synthesis of (1R,2S) and (lR,2R)-diastereoisomers 25 and 27" --- p.27 / Chapter II-5 --- "Synthesis of (1S,2R)-diastereoisomer 26 and Valiolamine" --- p.32 / Chapter II-6 --- "Comment on the Regio Chemistry of Nucleophilic Attack of 68, 65 and" --- p.85 / Chapter II-7 --- Results of Biological Test --- p.43 / Chapter III --- Conclusion --- p.46 / Chapter IV --- Experimental --- p.48 / Chapter V --- Reference --- p.80