β-Glucosidase zymograms of certain maize genotypes (nulls) do not show any activity bands after electrophoresis. We have shown that a chimeric lectin called β-glucosidase aggregating factor (BGAF) is responsible for the absence of β-glucosidase activity bands on zymograms. BGAF specifically binds to maize β-glucosidase isozymes Glu1 and Glu2 and forms large, insoluble complexes. Furthermore, we have previously shown that the N-terminal (Glu⁵⁰-Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions contain residues that make up the BGAF binding site on maize Glu1. However, sequence comparison between sorghum β-glucosidases (dhurrinases, Dhr1 and Dhr2), to which BGAF does not bind, and maize β-glucosidases, and an examination of the 3-D structure of Glu1 suggested that the BGAF binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²-Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹-Thr²⁹, together with C-terminal region Phe⁴⁶⁶-Ala⁵¹², affects the size of Glu1-BGAF complexes. To determine the importance of each region for binding, we determined the dissociation constants (K_d) of chimeric β-glucosidase-BGAF interactions. The results demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²-Thr⁸² on Glu1 for BGAF binding, we constructed chimeric Dhr2 (C-11, Dhr2 whose Val⁷²-Glu⁸² region was replaced with the Ile⁷²-Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²-Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding. We also constructed mutant β-glucosidases to identify and define the contribution of individual amino acids in the above three regions to BGAF binding. In the N-terminal region (Ile⁷²-Thr⁸²), critical region for BGAF binding, Glu1 mutants K81E and T82Y failed to bind BGAF in the gel-shift assay and their frontal affinity chromatography (FAC) profiles were essentially similar to that of sorghum β-glucosidase (dhurrinase 2, Dhr2), a non-binder, indicating that these two amino acids within Ile⁷²-Thr⁸² region are essential for BGAF binding. In the extreme N-terminal (Ser¹-Thr²⁹) and C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions, N481E [substitution of asparagine-481 with glutamic acid (as in Dhr)] showed lower affinity for BGAF, whereas none of the single amino acid substitutions in the Ser¹-Thr²⁹ region showed any effect on BGAF binding indicating that these regions play a minor role. To further confirm the importance of lysine-81 and threonine-82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four unique amino acids (isoleucine-72, asparagine-75, lysine-81, and threonine-82) of Glu1 in the peptide span Ile⁷²-Thr⁸² are required to impart BGAF binding ability to Dhr2. The sequence comparison among plant β-glucosidases supports the hypothesis that BGAF binding is specific to maize β-glucosidases because only maize β-glucosidases have threonine at position 82. / Ph. D.

β-glucosidases catalyze the hydrolysis of aryl and alkyl β-D-glucosides as well as glucosides with a carbohydrate moiety. The maize β-glucosidase isozymes Glu1and Glu2 hydrolyze a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc, while the sorghum β-glucosidase Dhr1 (dhurrinase-1) hydrolyzes exclusively its natural substrate dhurrin. For the expression of mature β-glucosidase isozymes Glu1 and Glu2 of maize and Dhr1 of sorghum, complementary DNAs were amplified by PCR and cloned into the expression vector pET-21a. Recombinant Glu1, Glu2 and Dhr1 enzymes were found to display activity towards the physiological substrates DIMBOAGlc and dhurrin, respectively, at levels similar to their native counterparts. It has been a subject of the subsequent studies by our lab and others to investigate what determines the aglycone specificity in β-glucosidases, and how β-glucosidases catalyze the hydrolysis of β-glycosidic bond between sugar and aglycone moieties. Molecular modeling techniques allowed to predict the substrate binding sites in Glu1 and Dhr1. Based on structural analysis of Glu1 and Dhr1, chimeric β-glucosidases (Glu1/Dhr1) were constructed by shuffling the C-terminal amino acids of Glu1 with the homologous region of Dhr1 to study the mechanism of substrate specificity. The resulting chimeric enzymes were characterized with respect to substrate specificity as well as kinetic, immunological, and electrophoretic properties. Shuffling a small portion of the C-terminal region altered the substrate specificity and improved by 2-4 fold the catalytic efficiency on other substrates in the chimeric β-glucosidases. These experiments showed that one or more of the 10 amino acid substitutions in the 30 amino acid long Dhr1 subdomain, 462SSGYTERFGIVYVDRENGCERTMKRSARWL491, plays a key role in dhurrin recognition and hydrolysis. To further investigate dhurrin recognition within this peptide region, two chimeric enzymes containing ⁴⁶²SSGYTERF⁴⁶⁹ and ⁴⁶⁶FAGFTERY⁴⁷³ Dhr1 peptides, respectively, were generated. The kinetic parameters indicated that Dhr1 peptide, ⁴⁶²SSGYTERF⁴⁶⁹, alone is sufficient to convert Glu1 to Dhr1 substrate specificity when it replaces the homologous peptide, ⁴⁶⁶FAGFTERY⁴⁷³, of Glu1. Maize β-glucosidases share high sequence similarities with Family 1 O-glucosidases. Therefore, these proteins are classified as retaining glycosyl hydrolases whose active site contains two glutamic acids (E) as the key catalytic residues, one as a general acid/base catalyst (E191) and the other as a nucleophile (E406). To confirm the identity and function of the acid/base catalyst E191, we have changed this residue to isosteric glutamine (Q) and aspartic acid (D) in both Glu1 and Glu2 isozymes by site-directed mutagenesis. The resulting mutant proteins were purified and their kinetic parameters (K_m, k_cat and k_i) were determined. The replacement of the acid/base catalyst E191 in the active site of maize β-glucosidase by Q and D resulted in inactivation of the enzyme. The kinetic analysis of the E191Q mutants showed that catalytic activity was reduced 200- and 110-fold towards ortho- and para-nitrophenyl- β-D-glucosides, respectively, when compared with the wild type enzyme. The E191D mutants showed no detectable activity towards any of the substrates tested. The back mutation of the E191Q mutants of the Glu1 and Glu2 isozymes to wild type restored full catalytic activity in both cases. These data indicate that E191 in maize β-glucosidases functions as an acid/base catalyst, and its function in catalysis cannot be performed by an isosteric residue such as glutamine or by a carboxyl group on a shorter side chain such as in aspartic acid. / Ph. D.

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.

>Magister Scientiae - MSc / The complete degradation of recalcitrant lignocellulose biomass into value-added products requires the efficient and synergistic action of lignocellulose degrading enzymes. This has resulted in a need for the discovery of new hydrolytic enzymes which are more effective than commonly used ones. β-xylosidases and β-glucosidases are key glycoside hydrolases (GHs) that catalyse the final hydrolytic steps of xylan and cellulose degradation, essential for the complete degradation of lignocellulose. Functional-based metagenomics has been employed successfully for the identification and discovery of novel GH genes from a metagenome library. Therefore, this approach was used in this study to increase the chances of discovering novel glycoside hydrolase genes from a horse manure metagenomic DNA library constructed in a previous study. Three fosmid clones P55E4, P81G1, and P89A4 exhibiting β-xylosidase activity were found to encode putative glycosyl hydrolases designated XylP55, XylP81, and BglP89. Amino acid sequence analysis revealed that XylP55, XylP81, and BglP89 are members of the GH43, GH39, and GH3 glycoside hydrolase families, respectively. Phylogenetic analysis of XylP81 and BglP89 indicated that these showed relatively low sequence similarities to other homologues in the respective GH families. The enzymes were expressed and purified, and only XylP81 and BglP89 were biochemically characterized. XylP81 (~58 kDa) and BglP89 (~84 kDa) both showed optimum activity at pH 6 and 50℃ and retained 100% residual activity at 55℃ after 1-hour indicating that they are moderately thermostable. XylP81 had high specific activity against 4-nitrophenyl-β-D-xylopyranoside (pNPX; 122 U/mg) with a KM value of 5.3 mM, kcat/KM of 20.3 s-1mM-1, and it showed enzyme activity against α-L-arabinofuranosidase, β-galactosidase, and β-glucosidase activity. BglP89 had a high specific activity for 4-nitrophenyl-β-D-glucopyranoside (pNPG; 133.5 U/mg) with a KM value of 8.4 mM, kcat/KM of 22 s-1mM-1 and also showed α-L-arabinofuranosidase, β-galactosidase, β-glucosidase, and low β-xylosidase activity. BglP89 also showed low hydrolytic activity on cellobiose, β-glucan, and lichenan indicating that it is a broad specificity β-glucosidase. XylP81 retained ~40% activity in the presence of 3 M xylose whilst BglP89 showed considerable glucose tolerance at 150 mM glucose and retained ~46% residual activity. This study reveals two metagenomic derived enzymes (β-xylosidase and β-glucosidase) showing characteristics that could make them potential candidates for lignocellulose biomass degradation in biotechnological and industrial applications.

The objectives of this study) are: (1) partial purification and characterization of rice β-glucosidase, (2) determination of the physiological role of the enzyme during rice germination, and (3) histochemical localization of the enzyme. The method for partial purification of the enzyme was based on that of Schliemann (1984), which included differential solubility, cryoprecipitation, and cation exchange chromatography. The enzymes were characterized with respect to their molecular weights, pI value, pli and temperature profile of activity and stability, activity in the presence of selected denaturants and organic’ solvents, substrate specificity, and inhibition by several known β-glucosidase inhibitors. To examine the physiological role of rice β-glucosidase, histochemical localization of the enzyme in dry seeds and application of inhibitors of the enzyme to the germinating seeds were carried out. The seeds were soaked in the presence or absence of β-glucosidase inhibitors, and the number of germinating seeds, growth and development of coleoptile and roots, and enzymatic activity of β-glucosidase and α-amylase were studied. To study histochemical localization of rice β-glucosidase, the chromogenic substrates were used. The substrates were incubated with cross and longitudinal sections of whole seeds and seedlings, tissue sections, protoplast and plastid preparations from 5-6-day-old coleoptiles. The development of the colors were observed under the light microscopes. Among the cation exchange chromatography fractions, two distinct peaks of oNPGase and pNPgase activity were found: fraction-1 (Fr-1) and fraction-2 (Fr-2) forms. It was found that the two forms of rice β-glucosidase are different with respect to susceptibility to denaturation by SDS, substrate specificity and some physico-chemical properties. Fr-1 is susceptible to denaturation by SDS, and catalyzes specifically the hydrolysis of several β-galactosides (pNPGal, X-gal, and 6-BNGal) but not gentiobiose and cellobiose, and is stable over pH range (4 to 10). Fr-2, on the other hand, is more resistant to denaturing agents, catalyzes the hydrolysis of gentiobiose and cellobiose, but not any of the β-galactosides mentioned above; it is relatively stable at pH 9, and less stable at high temperatures than Fr-1. Both Fr-1 and Fr-2 are 120 kD native dimers, made up of 60 kD monomers. In rice dry seeds, β-glucosidases were distributed in the aleurone layers and embryo parts. β-glucosidase inhibitors suppressed germination at the activation stage. The inhibitors Suppressed the expression of α-amylase and β-glucosidase during germination detected at the activity level. It is proposed, therefore, that the pre-existing f-glucosidase is involved in the regulation of availability and activity of a hormone (gibberellin) at the early step of germination that controls expression of hydrolytic enzymes such as α-amylase. In mature seeds, the Fr-1 is found mainly in the scutellum region and aleurone layers, while the Fr-2 form is in the axis of the embryo. In the seedling, the Fr-1 form is found in the scutellum, shoot and coleoptile, while the Fr-2 form is in the root. In young tissue of shoot and coleoptile, the enzyme is localized in the epidermis and vascular bundles. At the subcellular level it is localized to the plastids. / Ph. D.

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

As β-glicosidades são enzimas que catalisam a hidrólise de ligações glicosídicas. São amplamente encontradas na natureza em plantas, frutas e animais. Possuem diversas aplicações biotecnologicas podendo ser amplamente empregadas na indústria de alimentos e bebidas afim de melhorar a qualidade de aroma, sabor, coloração e viscosidade do produto. Este estudo apresenta o processo de imobilização de uma β-glicosidase comercial em suporte de quitosana e a obtenção de um derivado ativo e estável, para ser aplicado no processamento de vinhos aumentando a complexidade aromática de vinhos joven. A imobilizaçãpo foi realizada em suporte de quitosana, reticulado com glutaraldeído, atingindo 100% de eficiência na imobilização com 50mg de proteína por grama de suporte e 65% de atividade recuperada no derivado imobilizado. A imobilização além de contribuir para um maior controle do processo, alterou algumas características da β-glicosidase, a qual demonstrou manter uma atividade mais alta em faixas mais amplas de pH, quando comparada a enzima livre. A β-glicosidase imobilizada apresentou grande estabilidade podendo ser reutilizada por mais de 30 ciclos, mantendo sua atividade inicial. A aplicação da β-glicosidase no vinho foi realizada em batelada, por um tempo de 90 min, sob agitação. A análise por SPME/GC-MS revelou um aumento na concentração terpenos, quando comparada a amostras não tratadas. Houve um aumento na concentração de geraniol, citronelol, linalol e nerol. A aplicação da β-glicosidase foi bem sucedida, liberando os compostos aromáticos em um curto períuodo de tempo de contato. O processo de reutilização mostra que o biocatalisador imobilizado é uam ferramenta vantajosa para a indústria de bebidas. / β-glucosidases are enzymes that catalyze the hydrolysis of glycosidic bonds. They are widely found in nature at plants, fruits and animals. They have various biotechnological applications being largely used in food and beverage industry for the enhance the product viscosity, coloration, flavour and aroma qualities. This study presents a commercial β-glucosidase immobilization in chitosan support in order to obtain an active and stable derivative, enabling its application in winemaking, enhancing the aromatic complexity in young wines. The immobilization process was conducted in chitosana support, cross-linked with glutaraldehyde, reaching 100% efficiency in immobilization with 50 mg of protein per gram of support and 65% recovered activity in imobilized derived. The immobilization of the enzyme contributes to greater control of the process, changed some features of β-glucosidase, which proved to be more stable at pH changes when compared to free enzyme. Also the immobilized β-glucosidase showed great operational stability been reused for more than 30 cycles maintaining its initial activity. The application of β-glucosidase in the wine was held in batch for 90 minutes under stirring. The analyzis by SPME / GC-MS revelead a increasement in terpens concentration when compared to the sample without treatment. Was noticed a increase in geraniol, citronellol, linalool and nerol concentration. Apliccation of β-glucosidase was sucesfull, releasing aromatic compounds in contact for a short period of time. The reuses process showed that the immobilized biocatalyst is a advantageous tool for the beverage industry.

As β-glicosidades são enzimas que catalisam a hidrólise de ligações glicosídicas. São amplamente encontradas na natureza em plantas, frutas e animais. Possuem diversas aplicações biotecnologicas podendo ser amplamente empregadas na indústria de alimentos e bebidas afim de melhorar a qualidade de aroma, sabor, coloração e viscosidade do produto. Este estudo apresenta o processo de imobilização de uma β-glicosidase comercial em suporte de quitosana e a obtenção de um derivado ativo e estável, para ser aplicado no processamento de vinhos aumentando a complexidade aromática de vinhos joven. A imobilizaçãpo foi realizada em suporte de quitosana, reticulado com glutaraldeído, atingindo 100% de eficiência na imobilização com 50mg de proteína por grama de suporte e 65% de atividade recuperada no derivado imobilizado. A imobilização além de contribuir para um maior controle do processo, alterou algumas características da β-glicosidase, a qual demonstrou manter uma atividade mais alta em faixas mais amplas de pH, quando comparada a enzima livre. A β-glicosidase imobilizada apresentou grande estabilidade podendo ser reutilizada por mais de 30 ciclos, mantendo sua atividade inicial. A aplicação da β-glicosidase no vinho foi realizada em batelada, por um tempo de 90 min, sob agitação. A análise por SPME/GC-MS revelou um aumento na concentração terpenos, quando comparada a amostras não tratadas. Houve um aumento na concentração de geraniol, citronelol, linalol e nerol. A aplicação da β-glicosidase foi bem sucedida, liberando os compostos aromáticos em um curto períuodo de tempo de contato. O processo de reutilização mostra que o biocatalisador imobilizado é uam ferramenta vantajosa para a indústria de bebidas. / β-glucosidases are enzymes that catalyze the hydrolysis of glycosidic bonds. They are widely found in nature at plants, fruits and animals. They have various biotechnological applications being largely used in food and beverage industry for the enhance the product viscosity, coloration, flavour and aroma qualities. This study presents a commercial β-glucosidase immobilization in chitosan support in order to obtain an active and stable derivative, enabling its application in winemaking, enhancing the aromatic complexity in young wines. The immobilization process was conducted in chitosana support, cross-linked with glutaraldehyde, reaching 100% efficiency in immobilization with 50 mg of protein per gram of support and 65% recovered activity in imobilized derived. The immobilization of the enzyme contributes to greater control of the process, changed some features of β-glucosidase, which proved to be more stable at pH changes when compared to free enzyme. Also the immobilized β-glucosidase showed great operational stability been reused for more than 30 cycles maintaining its initial activity. The application of β-glucosidase in the wine was held in batch for 90 minutes under stirring. The analyzis by SPME / GC-MS revelead a increasement in terpens concentration when compared to the sample without treatment. Was noticed a increase in geraniol, citronellol, linalool and nerol concentration. Apliccation of β-glucosidase was sucesfull, releasing aromatic compounds in contact for a short period of time. The reuses process showed that the immobilized biocatalyst is a advantageous tool for the beverage industry.

As β-glicosidades são enzimas que catalisam a hidrólise de ligações glicosídicas. São amplamente encontradas na natureza em plantas, frutas e animais. Possuem diversas aplicações biotecnologicas podendo ser amplamente empregadas na indústria de alimentos e bebidas afim de melhorar a qualidade de aroma, sabor, coloração e viscosidade do produto. Este estudo apresenta o processo de imobilização de uma β-glicosidase comercial em suporte de quitosana e a obtenção de um derivado ativo e estável, para ser aplicado no processamento de vinhos aumentando a complexidade aromática de vinhos joven. A imobilizaçãpo foi realizada em suporte de quitosana, reticulado com glutaraldeído, atingindo 100% de eficiência na imobilização com 50mg de proteína por grama de suporte e 65% de atividade recuperada no derivado imobilizado. A imobilização além de contribuir para um maior controle do processo, alterou algumas características da β-glicosidase, a qual demonstrou manter uma atividade mais alta em faixas mais amplas de pH, quando comparada a enzima livre. A β-glicosidase imobilizada apresentou grande estabilidade podendo ser reutilizada por mais de 30 ciclos, mantendo sua atividade inicial. A aplicação da β-glicosidase no vinho foi realizada em batelada, por um tempo de 90 min, sob agitação. A análise por SPME/GC-MS revelou um aumento na concentração terpenos, quando comparada a amostras não tratadas. Houve um aumento na concentração de geraniol, citronelol, linalol e nerol. A aplicação da β-glicosidase foi bem sucedida, liberando os compostos aromáticos em um curto períuodo de tempo de contato. O processo de reutilização mostra que o biocatalisador imobilizado é uam ferramenta vantajosa para a indústria de bebidas. / β-glucosidases are enzymes that catalyze the hydrolysis of glycosidic bonds. They are widely found in nature at plants, fruits and animals. They have various biotechnological applications being largely used in food and beverage industry for the enhance the product viscosity, coloration, flavour and aroma qualities. This study presents a commercial β-glucosidase immobilization in chitosan support in order to obtain an active and stable derivative, enabling its application in winemaking, enhancing the aromatic complexity in young wines. The immobilization process was conducted in chitosana support, cross-linked with glutaraldehyde, reaching 100% efficiency in immobilization with 50 mg of protein per gram of support and 65% recovered activity in imobilized derived. The immobilization of the enzyme contributes to greater control of the process, changed some features of β-glucosidase, which proved to be more stable at pH changes when compared to free enzyme. Also the immobilized β-glucosidase showed great operational stability been reused for more than 30 cycles maintaining its initial activity. The application of β-glucosidase in the wine was held in batch for 90 minutes under stirring. The analyzis by SPME / GC-MS revelead a increasement in terpens concentration when compared to the sample without treatment. Was noticed a increase in geraniol, citronellol, linalool and nerol concentration. Apliccation of β-glucosidase was sucesfull, releasing aromatic compounds in contact for a short period of time. The reuses process showed that the immobilized biocatalyst is a advantageous tool for the beverage industry.

Thesis (MSc. (Biochemistry)) --University of Limpopo, 2016 / The ever increasing cost of fossil-based fuels and the accompanying concerns about their impact on the environment is driving research towards clean and renewable sources of energy. Bioethanol has the potential to be a replacement for liquid transportation fuels. In addition to its near zero nett carbon dioxide emissions, bio-ethanol has a high energy to weight ratio and can easily be stored in high volumes. To produce bioethanol at economically competitive prices, the major cost in the production process needs to be addressed. The addition of enzymes to hydrolyse the lignocellulosic fraction of the agricultural waste to simple sugars is considered to be the major contributor to high production cost. A consolidated bioprocess (CBP) which ideally combines all the steps that are currently accomplished in different reactors by different microorganisms into a single process step would be a more economically feasible solution. In this study the potential of yeast hybridization with a CBP approach was used. In order to evaluate the reduction or elimination of the addition of cellulolytic and hemi-cellulolytic enzymes to the ethanol production process. High cellobiohydrolase I secreting progeny from hybridization of an industrial bioethanol yeast strain, S. cerevisiae M0341, and a laboratory strain S. cerevisiae Y294 were isolated. In order to determine if this characteristic was specific to cellobiohydrolase I secretion, these strains were evaluated for their ability to secrete other relevant recombinant hydrolase enzymes for CBP-based ethanol production. A total of seven S. cerevisiae strains were chosen from a progeny pool of 28 supersecreting hybrids and reconstructed to create two parental strains; S. cerevisiae M0341 and S. cerevisiae Y294, together with their hybrid segregants strains H3M1, H3M28, H3H29, H3K27 and H3O23. Three episomal plasmids namely pNS201, pNS202 and pNS203 were constructed; these plasmids together with two already available plasmids, namely pRDH166 and pRDH182 contained genes for different reporter enzymes, namely β-glucosidase I, xylanase II, endoglucanase lll, cellobiohydrolase l and α-glucuronidase. To allow for selection of the episomal plasmids, homologous recombination was used to replace the functional URA3 gene of selected strains, with the non-functional ura3 allele from the Y294 strain. Enzyme activity was used as an indicator of the amount of enzyme secreted. Fermentation studies in a bioreactor were used to determine the metabolic burden imposed on the segregants expressing the cellobiohydrolase at high levels. In addition all segregants were tested for resistance to inhibitors commonly found in pre-treated lignocellulosic material. The M28_Cel7A was found to be the best secretor of Cel7A (Cellobiohydrolase l); however it seems as though this phenomenon imposes a significant metabolic burden on the yeast. The supersecreting hybrid strains cannot tolerate lignocellulosic inhibitors at concentrations commonly produced during pretreatment / The National Research Foundation - Renewable Energy Scholarship (NRF-RSES)