The increasing level and dispersion of toxic chemicals in the environment has stimulated a need for accurate methods capable of detecting and quantifying the activity of toxicants. The primary objective of this study was to determine, through in vitro tests, the potential of three purified enzymes: α-chymotrypsin, acid phosphatase, and carbonic anhydrase for use in the early assessment of toxicants at environmentally significant levels. Activities of α-chymotrypsin and acid phosphatase were measured spectrophotometrically, while carbonic anhydrase activities were determined through monitoring a pH change. The chemical agents investigated included several heavy metals, common herbicides and pesticides, and various environmentally significant anions. In addition, several techniques were explored to amplify enzyme response to chemical agents. The results of the study demonstrated that α-chymotrypsin did not significantly respond to cadmium, nickel, or 2,4-D, and consequently, does not appear to be useful in indicating potential toxicity problems associated with these agents. The acid phosphatase enzyme system appears to be useful in signaling the presence of low levels of certain anions (fluoride at 0.45 mg/ℓ and nitrate at 7.3 mg/ℓ), but does not appear to have potential for detecting toxic activity due to cadmium, nickel, or 2,4-D. Enzyme inhibition induced by fluoride at 37°C was not altered by changing the assay temperature to 50°C. The results of the experiments with carbonic anhydrase show that the enzyme does not appear to be affected by cadmium or nickel ions. However, enzyme activity was inhibited by fluoride (4.5 mg/ℓ), sulfide (0.5 mg/ℓ), and nitrate (73 mg/ℓ). Enzyme inhibition was also induced by 10 mg/ℓ of atrazine, malathion, or carbaryl, and 150 to 500 mg/ℓ of 2,4-D. Inhibitory effects induced by sulfanilamide appeared to be slightly enhanced by the addition of Cd²⁺, Ni²⁺, or Zn²⁺ cations. These findings, although preliminary, suggest that carbonic anhydrase demonstrates potential for signaling the presence of anions, and appears to be useful in indicating potential toxicity problems due to pesticides and herbicides. / Master of Science

Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The distinctive varietal flavour of wines is a combination of absolute and relative concentrations of chemical compounds. Volatile compounds are responsible for the odour of wine and non-volatiles cause the sensation of flavour. Accompanying these senses, a third, tactile, sense of ‘mouth-feel’ is recognizable. This forms the complete organoleptic quality of wine. Several hundred different compounds are simultaneously responsible for the odour release in wine, and since there is no real character impact compound, the aroma of wine can be described as a delicate balance of all these compounds. One of the most important groups of volatiles is the monoterpenes, which play a role in both aroma and flavour. This is especially significant for the Muscat varieties, but these flavour compounds are also present in other non-muscat grape varieties, where they supplement the varietal aroma. Monoterpenes occur in wine as free, volatile and odorous molecules, as well as flavourless non-volatile glycosidic complexes. The latter slowly releases monoterpenes by acidic hydrolysis, but the impact on varietal aroma is considered insufficient for wines that are consumed young. It is therefore important to supplement the release mechanism, in order to enhance the varietal aroma of the wine. The enzymatic hydrolysis mechanism functions in two successive steps: firstly, depending on the precursor, the glycosidic linkage is cleaved by α-L-arabinofuranosidase, α-L-rhamnosidase, β-D-xylosidase or β-D-apiosidase. The second step involves the liberation of the monoterpene alcohol by a β-glucosidase. This enzymatic hydrolysis does not influence the intrinsic aromatic characteristics of the wine, as opposed to acid hydrolysis. Pectolytic enzymes play an important role in cell elongation, softening of tissue and decomposition of plant material. These enzymes are used to improve juice yields, release colour and flavour compounds from grape skins, as well as improve clarification and filterability. Pectolytic enzymes work synergistically to break down pectins in wine. Protopectinase produce water-soluble and highly polymerised pectin substances from protopectin, it acts on non-methylated galacturonic acid units. Pectin methylesterase split methyl ester groups from the polygalacturonic chain. Polygalacturonase break down the glycosidic links between galacturonic acid units. Pectin and pectate lyases have a β-eliminative attack on the chain and it results in the formation of a double bond between C4 and C5 in the terminal residues. From the above it can be seen that enzymes play a pivotal role in the winemaking process. Unfortunately, in winemaking a lot of factors can influence the effects of enzymes. One possible factor in the wine medium is the presence of acidprotease, from yeast and/or fungal origin. This type of enzyme utilizes other enzymes as substrates and renders them useless. Pure enzyme preparations were used to study the interactions of a yeast acid-protease and a report activity (β-glucosidase) in vitro. A bottled wine and a buffer were used as in vitro conditions. Enzyme assays were performed to determine the relative activity over a number of days. The results indicated that even though both enzymes showed activity in both the media, the yeast protease did not have any significantly affect on the report activity. Subsequently wine was made from Sauvignon blanc grapes, with varying enzyme preparation additions. Enzyme assays were performed during the fermentation; and chemical, as well as sensory analysis were done on the stabilized wine. The results confirmed that the yeast protease did not have any significant affect on the report activity in these conditions. The protease’s inability to affect the report activity seems unlikely due to the fact that it is active at a low pH range and has been suggested as the only protease to survive the fermentation process. It seems possible that a winerelated factor, possibly ethanol, is responsible. Thus it seems that yeast protease does not threaten the use of commercial enzymes in the winemaking process in any significant way. Future work would entail more detailed enzyme studies of interactions between protease, both from yeast and fungal origin, and other report activities in specified conditions. The degradation capability could be directed towards unwanted enzyme activities that cause oxidation and browning of the must. The characterization of interactions between protease and β-glucosidase activities may hold key to producing wines with enhanced aroma and colour potential, as well as the elimination of unwanted enzyme activities. / AFRIKAANSE OPSOMMING: Die herkenbare kultivar karakter van wyn is ‘n kombinasie van absolute en relatiewe konsentrasies van verskeie chemiese komponente. Vlugtige komponente is verantwoordelik vir die geur, of aroma, van wyn en die nie-vlugtige komponente veroorsaak die sensasie van smaak. ‘n Derde, fisiese sensasie, die ‘mondgevoel’, is ook herkenbaar. Dit vorm die omvattende organoleptiese kwaliteit van die wyn. ‘n Paar honderd verskillende komponente is gelyktydig verantwoordelik vir die aroma vrystelling in wyn en omdat daar geen werklike karakter ‘impak’ komponent is nie, kan die aroma van wyn beskryf word as ‘n delikate balans van al die betrokke komponente. Een van die mees belangrike groepe vlugtige komponente is die monoterpene wat ‘n rol speel in beide aroma en smaak. Dit is veral belangrik by Muskaat kultivars, maar hierdie aroma komponente is ook teenwoordig in niemuskaat druif kultivars, waar hulle bydra tot die kultivar karakter en aroma. Monoterpene kom in wyn voor as vry, vlugtige en aromatiese molekules en in geurlose, nie-vlugtige glikosidies-gebonde komplekse. Die gebonde vorm word stadig vrygestel deur ‘n suurhidrolise, maar dit word as onvoldoende beskou vir wyne wat vroeg gedrink word. Dit is dus belangrik dat die vrystelling van geurstowwe verhoog word om die kultivar karakter van die wyn te versterk. Die ensiematiese hidrolise proses behels twee opeenvolgende stappe: eerstens, afhangende van die aard van die voorloper, word die glikosidiese verbinding deur α-L-arabinofuranosidase, α-Lramnosidase, β-D-xilosidase, of β-D-apiosidase gebreek. In die tweede stap word die monoterpeen-alkohol deur β-glukosidase vrygestel. Hierdie ensiematiese afbraak proses verander nie die intrinsieke aromatiese kenmerke van die wyn, soos met suurhidrolise die geval is nie. Pektolitiese ensieme speel ‘n fundamentele rol in selverlenging, sagwording en afbraak van plant materiaal. Hierdie ensieme word gebruik om sap opbrengs te verhoog, aroma en smaak komponente vry te stel uit die doppe, asook om sapverheldering en filtrasie te verbeter. Die pektolitiese ensieme werk op ‘n sinergistiese wyse om pektien in wyn af te breek. Protopektinase produseer wateroplosbare en hoogs gepolimeriseerde pektien uit protopektien, slegs uit niegemetileerde galakturoonsuur eenhede. Pektien metielesterase verwyder metielester groepe van die poligalakturoonsuurketting. Die glikosidiese bindings tussen galakturoonsuur eenhede word deur poligalakturonase afgebreek. Pektien- en pektaat-liase het ‘n β-eliminasie aanslag op die ketting en as gevolg daarvan word dubbelbindings tussen C4 en C5 in die terminale residue gevorm. Vanuit bogenoemde is dit dus duidelik dat ensieme ‘n kardinale rol speel in die wynbereidingsproses. Ongelukkig is daar ‘n verskeidenhied van faktore wat die werking van ensieme in die wynbereidingsproses kan beïnvloed. Een moontlike faktor is die teenwoordigheid van ‘n suur-protease, van fungisidiese en/of gis oorsprong, in die wynmedium, omdat dit ander ensieme as substraat kan benut en degradeer. Suiwer ensiem preparate is gebruik om die ensiem interaksie tussen ‘n gis suur-protease en ‘n verslag aktiwiteit (β-glukosidase) in vitro te ondersoek. ‘n Gebotteleerde wyn en ‘n buffer is gebruik om die in vitro kondisies na te boots. Relatiewe ensiem aktiwiteit is ontleed oor ‘n aantal dae. Beide die ensieme het aktiwiteit getoon in die media, maar gis protease het geen statisties beduidende invloed gehad op die aktiwiteit van die verslag ensiem nie. Daaropvolgend is wyn berei van Sauvignon blanc druiwe, met verskillende ensiempreparaat toevoegings. Die ensiemaktiwiteit is deurlopend tydens fermentasie gemeet. Na afloop van stabilisasie is chemiese, sowel as sensoriese ontledings op die wyn gedoen. Die resultate het bevestig dat gis protease, onder hierdie kondisies, geen beduidende invloed op die verslag aktiwiteit gehad het nie. Die protease se onvermoë om die verslag aktiwiteit beduidend te beinvloed blyk onwaarskynlik aangesien die suurprotease aktief is by lae pH vlakke en dit as die enigste protease voorgestel is wat die fermentasie proses kan oorleef. Dit blyk asof ‘n wyn-verwante faktor, moontlik etanol, hiervoor verantwoordelik kan wees. Dus hou protease geen gevaar in vir die gebruik van kommersiële ensieme in wynbereiding nie. Navorsing kan in die toekoms fokus op meer gedetailleerde ensiem interaksie studies tussen protease en ander ensiem aktiwiteite, in gespesifiseerde kondisies. Die degradasie kapasiteit kan moontlik aangewend word om ongewenste ensiem aktiwiteite, wat byvoorbeeld oksidasie en verbruining veroorsaak, te verminder. Die karakterisering van die interaksies tussen protease en β-glukosidase kan dus die sleutel wees tot die produksie van wyne met verhoogde aroma potensiaal, asook die eliminasie van ongewenste ensiematiese aktiwiteite.

Thesis (Msc (Viticulture and Oenology. Institute for Wine Biotechnology))--University of Stellenbosch, 2009. / Monoterpenes are important aroma compounds in certain grape varieties such as Muscat, Gewürztraminer and Riesling and are present as either odourless, glycosidically bound complexes or as free aromatic monoterpenes. These complexes occur as monoglucosides or, when present as diglycosides, most commonly as 6-O-α-L-arabinofuranosyl-β-D-glucopyranosides of mainly linalool, geraniol, nerol and citronellol. The release of monoterpenes from non-volatile glycosidically bound precursors occurs either by acid hydrolysis or enzymatic hydrolysis. High temperature acid hydrolysis causes a rearrangement of the monoterpene aglycones and a decrease in the aroma and changes in the aromatic characteristics of monoterpenes and is therefore not suitable. Enzymatic hydrolysis does not modify the monoterpene aglycones and can be an efficent method to release potentially volatile monoterpenes. α-L-arabinofuranosidase and β-glucosidase are important enzymes responsible for the liberation of monoterpene alcohols from their glycosides. Glycosidases from Vitis vinifera and Saccharomyces cerevisiae are severely inhibited by winemaking conditions and this leads to unutilized aroma potential, while commercial preparations of aroma liberating enzymes are crude extracts that often have unwanted and unpredictable side effects. It is therefore of interest to investigate alternative measures to release glycosidically bound monoterpenes to increase the floral aroma of wine without side activities that impact negatively on wine. Heterologous α-L-arabinofuranosidases and β-glucosidases have previously been expressed in S. cerevisiae and these studies have evaluated and found increased glycosidic activities against both natural and synthetic substrates. In this study, we expressed the Aspergillus awamori α-L-arabinofuranosidase (AwAbfB) in combination with either the β-glucosidases Bgl2 from Saccharomycopsis fibuligera or the BglA from Aspergillus kawachii in the industrial yeast strain S. cerevisiae VIN13 to facilitate the sequential enzymatic hydrolysis of monoterpene diglycosides. Enzyme assays and GC-FID (Gas Chromatography with a Flame Ionization Detector) results show a significant increase in the amount of free monoterpene concentrations under winemaking conditions in the strain co-expressing the AwAbfB and the Bgl2. The increases in free monoterpene levels obtained were similar to those obtained with a commercial enzyme preparation, LAFAZYM AROM. Sensorial evaluation confirmed the improvement in the wine aroma profile, particularly the floral character. This yeast strain permits a single culture fermentation which improves the sensorial quality and complexity of wine. Further investigations on the factors influencing the stability and reactivity of monoterpenes during alcoholic fermentation are needed.

Made available in DSpace on 2014-11-10T11:09:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-05-29Bitstream added on 2014-11-10T11:58:01Z : No. of bitstreams: 1 000789630_20141229.pdf: 409322 bytes, checksum: ffbf7ab412d7ca57518c2014df4f7a52 (MD5) Bitstreams deleted on 2015-01-05T11:00:54Z: 000789630_20141229.pdf,Bitstream added on 2015-01-05T11:01:50Z : No. of bitstreams: 1 000789630.pdf: 2048959 bytes, checksum: 453ad5bd7d63b78e68c4bf76a6845403 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Este trabalho visou dar tratamento tecnológico a extratos enzimáticos ricos em endoglucanases obtidos por cultivo em estado sólido (CES) do fungo termofílico Myceliophtora thermophila M77, através da secagem por spray-drying. A fim de garantir a estabilidade das enzimas durante um período de armazenamento, o extrato enzimático bruto (EEB) foi seco em spray-dryer sendo investigados os parâmetros operacionais e os adjuvantes de secagem para garantir a maior retenção da atividade enzimática após a secagem. Os ensaios foram divididos entre discriminação do adjuvante que proporcionasse maior proteção às enzimas, otimização das condições de secagem, armazenamento do EEB e dos pós secos, avaliação do efeito da secagem sobre as propriedades físico-químicas das endoglucanases e morfologia dos pós. Utilizou-se de planejamentos estatísticos para analisar os efeitos das variáveis temperatura de saída do ar de secagem, vazão de alimentação de solução, proporção entre o EEB e adjuvante, e teor de sólidos na formulação, sendo as respostas retenção de atividade enzimática e umidade dos pós. Nos ensaios de discriminação foi selecionado o adjuvante goma arábica. Em seguida, nos ensaios para otimização das condições de secagem, foi identificado menor vazão de alimentação e menores temperaturas de saída do ar de secagem para maiores retenções de atividade enzimática e maiores temperaturas e menores vazões para menores teores de umidade dos pós, o teor de sólidos revelou-se não significativo. O pó obtido nas condições mais favorável e menos favorável para a retenção da atividade enzimática foi armazenado à temperatura ambiente e sob refrigeração e a atividade enzimática determinada quinzenalmente. A análise estatística revelou não haver diferença na retenção da atividade enzimática após longos períodos para ambas as condições de secagem selecionadas e alternativas de armazenamento... / This work aimed to provide technological treatment to enzymatic extract rich in endoglucanase, obtained by solid state culture using the fungus Myceliophtora thermophila M77, through spray-drying. Operational parameters and adjuvants were varied in order to obtain stable enzymes with high activities during storage. The experiments were divided into adjuvant discrimination, optimization of the drying conditions, storage of the raw enzymatic extract (REE) and powders, evaluation of drying on the physical-chemical characteristics of the endoglucanase, and powder morphology. Statistical experimental designs were used to evaluate the effect of the variables exit air temperature, solution flow rate, proportion REE/adjuvant, and total solid content on the enzyme activity retention and powder moisture content. The adjuvant Arabic gum was selected from the discrimination assays. From the optimization experiments, it was determined that the lower flow rate and air exit temperature resulted in the highest enzyme activity retention, while low temperature and solution flow rate resulted in the lowest retention. The total solid concentration was not statistically significant. The powders produced in the Best and in the worst drying conditions were stored at room temperature and under refrigeration and the enzymatic activity was measured at 15 days intervals. The statistical analysis did not show any difference in the enzymatic activity retention after long periods of storage for both drying conditions and both storage alternatives. The influence of temperature and pH on the enzyme activity was also investigated before and after the drying process, and it was noticed that these physical-chemical properties were not affected by the adjuvants and by the drying process. The morphology of the powders, obtained using the inert gas adsorption method, indicated that the powder obtained under the worst drying condition presented specific superficial area twice ...

Made available in DSpace on 2014-12-02T11:16:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-11-29Bitstream added on 2014-12-02T11:21:22Z : No. of bitstreams: 1 000791747_20160107.pdf: 391327 bytes, checksum: 7bae48548e730d96c10d4d65576e4e26 (MD5) Bitstreams deleted on 2016-01-12T18:57:18Z: 000791747_20160107.pdf,. Added 1 bitstream(s) on 2016-01-12T18:58:35Z : No. of bitstreams: 1 000791747.pdf: 1679458 bytes, checksum: cbef661cf8e1e32aaff5b1b1acd034bc (MD5) / Devido à ampliação das aplicações industriais das celulases e hemicelulases, é crescente o interesse na produção destas enzimas por micro-organismos através de processos que promovam altos rendimentos a baixo custo. Assim, o cultivo de fungos filamentosos por fermentação em estado sólido (FES) utilizando-se resíduos lignocelulósicos como substratos tem sido frequentemente citado na literatura. Os fungos termofílicos tem se destacado neste sentido, por produzirem enzimas geralmente mais ativas e estáveis sob altas temperaturas, uma vantagem do ponto de vista das aplicações industriais. No presente trabalho, foram isolados 32 fungos termofílicos do ambiente a partir de amostras de silagem, compostagem e outros materiais orgânicos. Uma triagem inicial dos melhores produtores foi feita pelo cultivo dos isolados, por FES, em mistura de bagaço de cana-de-açúcar e farelo de trigo (1:1 p/p) como substratos, por 96h. A partir destes cultivos foram pré-selecionados 7 isolados, os quais foram então cultivados por FES em diferentes misturas de resíduos agro-industriais, por 96h, a fim de se avaliar a influência dos mesmos na produção das enzimas, buscando-se maiores rendimentos. Foram então selecionados para dar continuidade ao trabalho os isolados que mais se destacaram na produção das enzimas: Myceliophthora thermophila JCP 1-4 (endoglucanase, -glicosidase, xilanase e avicelase) e DABM 11/45 (-glicosidase, xilanase e avicelase). A influência do tempo de cultivo na produção das enzimas pelos dois isolados selecionados foi avaliada e as enzimas foram caracterizadas físico-quimicamente. O extrato enzimático produzido por M. thermophila, por FES, em mistura de bagaço de cana-de-açúcar e farelo de soja (1:1 p/p) foi utilizado para a sacarificação de bagaço de cana in natura e pré-tratado com ozônio. Foram ainda realizados ensaios de sacarificação com enzimas comerciais, para fins comparativos. Os bagaços in . / Due to the expansion of industrial applications of cellulases and hemicellulases, there is increasing interest in the production of these enzymes by micro-organisms through processes that promote high yields at low cost. Thus, the cultivation of filamentous fungi by solid state fermentation (SSF) using lignocellulose residues as substrates has been frequently cited in the literature. The thermophilic fungi has excelled in this regard, since they produce enzymes usually more active and stable under high temperatures, an advantage from the point of view of industrial applications. In this work, 32 thermophilic fungi were isolated from the environment, from samples of silage, composting and other organic materials. An initial screening of the best producers was made by cultivation of isolates, by SSF, in a mixture of sugar cane bagasse and wheat bran (1:1 w/w) as substrates, for 96h. From these cultivations 7 isolates were pre-selected, which were then cultured by SSF in different mixtures of agro-industrial residues, for 96h, in order to assess the influence of these substrates in enzymes production, seeking higher yields. Then, were selected to give continuity to the work the isolates that stood out in the production of enzymes: Myceliophthora thermophila JCP 1-4 (endoglucanase, -glucosidase, xylanase and avicelase) and DABM 11/45 (-glucosidase, xylanase and avicelase). The influence of the time on the enzymes production by the two selected isolates was assessed and the enzymes were characterized physico-chemically. The enzymatic extract produced by M. thermophila, by FES, in a mixture of sugar cane bagasse and soybean meal (1:1 w/w) was used for the saccharification of in natura and pre-treated with ozone sugarcane bagasse. Saccharification assays were also performed with commercial enzymes, for comparative purposes. The in natura and ozonized bagasse were characterized regarding the content of cellulose, hemicellulose and lignin ...

Neste trabalho, são descritos os detalhes da montagem de um sistema de cultivo microbiano em estado sólido, cujo elemento principal é um biorreator de tambor rotativo em escala de bancada, com casco em vidro refratário com aproximadamente 6,2 litros de volume. A montagem do equipamento exigiu estudos para a definição dos seguintes aspectos: geometria do reator; controle de frequência e período de agitação; umidificação, aquecimento e controle do fluxo do ar injetado no sistema; mistura do meio de cultivo; medição e controle da temperatura do meio; retirada de amostras. O sistema desenvolvido foi avaliado, tendo como processo fermentativo modelo o cultivo de Aspergillus niger T0005/007-2, microrganismo produtor de enzimas pectinolíticas. Os testes foram feitos em um meio contendo farelo de trigo como suporte sólido, avaliando-se a influência de três parâmetros principais agitação, massa de meio de cultivo e temperatura do meio sobre o crescimento celular e a produção de endo-poligalacturonase (PG) por A. niger. Três formas de agitação do meio sólido foram comparadas: sem agitação; agitação a 1 rpm por 5 minutos a cada duas horas; agitação a 1 rpm por 1 hora e 55 minutos a cada duas horas. A segunda forma de agitação levou ao melhor crescimento celular, 81 mg.g-1, e a atividade de endo-PG da ordem de 80 U.g-1, semelhante ao estimado com o sistema estático. Com a terceira forma de agitação, aparentemente houve dano ao micélio fúngico e, com isso, resultados inferiores foram alcançados. A avaliação do efeito da massa de meio de cultivo sobre o processo foi feita com cargas crescentes de meio que resultaram na ocupação de 30, 45 e 60 % do volume útil do reator. Quanto ao crescimento celular, o melhor resultado foi alcançado com a menor carga de meio, enquanto que a carga intermediária resultou no mais alto título enzimático final, 107,2 U.g-1. Nos experimentos sobre a influência da temperatura sobre o cultivo de A. niger, a maior atividade enzimática (80,6 U.g-1) foi obtida numa condição de trabalho que permitiu que a temperatura do meio atingisse valores da ordem de 45ºC. Com o meio mantido a 30ºC, a atividade enzimática máxima foi substancialmente mais baixa, 46,4 U.g-1. Nos testes realizados com o processo modelo, não se verificou uma clara influência positiva da agitação sobre a produção de endo-PG. Entretanto, a partir dos experimentos com temperatura controlada, é possível sugerir que a formação de endo-PG é favorecida por uma condição de estresse para o microrganismo, no caso representado por temperaturas do meio acima de 40ºC, visto que a temperatura ideal de crescimento de A. niger encontra-se na faixa de 28 a 34ºC. Este resultado, que discorda do que é genericamente descrito na literatura especializada sobre cultivos em estado sólido, é um exemplo da importância de dispor-se de um biorreator de tambor rotativo de pequena escala como equipamento básico para a realização de estudos fundamentais a respeito deste tipo de processo. Adicionalmente, o fato de o corpo do reator ser construído em vidro permite a visualização do espaço interno do tambor rotativo e observar o efeito da agitação sobre o meio de cultivo. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-06-02T17:41:21Z No. of bitstreams: 1 Dissertacao Tomas Augusto Polidoro.pdf: 1493269 bytes, checksum: 0ed8a1384e54fe0a00f47be237707cbf (MD5) / Made available in DSpace on 2014-06-02T17:41:21Z (GMT). No. of bitstreams: 1 Dissertacao Tomas Augusto Polidoro.pdf: 1493269 bytes, checksum: 0ed8a1384e54fe0a00f47be237707cbf (MD5) / In this work, details on the assembling of a solid state cultivation system, whose main component is a bench scale rotating-drum bioreactor, are described. The bioreactor was built in refractory glass and has an approximate volume of 6.2 liters. To assemble the equipment, the following aspects have been studied: geometry of the bioreactor; control of frequency and period of agitation; moistening, warming and controlling of the inlet air flux; mixing of cultivation medium; determination and control of medium temperature; sample withdrawn. The developed system was evaluated, being the cultivation of the pectinolytic enzymeproducing microorganism Aspergillus niger T0005/007-2 used as the model process. The fermentative tests were carried out in a medium containing wheat-straw as solid support and were used to evaluate the effects of three main parameters agitation, mass of cultivation medium and medium temperature on the cell growth and the production of endopolygalacturonase (endo-PG) by A. niger. For agitation of the solid medium, three modes were compared: no agitation; agitation of 1 rpm for 5 minutes each 2 hours; agitation for 1 hour and 55 minutes, each 2 hours. The second mode of agitation led to the best cell growth, 81 mg.g-1, and to endo-PG activity of approximately 80 U.g-1, similar to that obtained with the static system. With the third mode of agitation occurred, apparently, some damage to fungus mycelium and inferior results were achieved. The evaluation of the mass of cultivation medium on the process was done by loading the reactor with masses that result in the occupation of 30, 45 and 60% of the working volume of the bioreactor. With respect to the cell growth, the best result was attained with the smallest load of medium, whereas the intermediate load resulted in the highest endo-PG activity, 107.2 U.g-1. In the experiments on the influence of the temperature on the cultivation of A. niger, the largest endo-PG activity (80.6 U.g-1) was obtained in a process condition that allowed that the temperature reached values close to 45ºC. When the medium temperature was controlled at 30ºC, the endo-PG activity was substantially lower, 46.4 U.g-1. In the tests with the model process, no clearly positive influence of agitation on the production of endo-PG was observed. From the temperature-controlled experiments however, it is possible to suggest that endo-PG formation is favored by a stress condition for the microorganism, represented in this case by medium temperatures over 40ºC, since the optimal temperatures for A. niger growth is found in the range of 28 to 34ºC. This result disagrees from what is generically described in the specialized literature for the solid state cultivations, being this fact an example of the importance of having a small-scale rotating-drum bioreactor as a basic equipment for fundamental studies on that type of fermentative process. Additionally, the fact of the reactor body being built on glass allows the inspection of the internal space of the rotating drum and to observe the effects of agitation on the cultivation medium.

Orientador: João Cláudio Thoméo / Banca: Gustavo Orlando Bonilla Rodriguez / Banca: Izabela Dutra Alvin / Resumo: Este trabalho visou dar tratamento tecnológico a extratos enzimáticos ricos em endoglucanases obtidos por cultivo em estado sólido (CES) do fungo termofílico Myceliophtora thermophila M77, através da secagem por spray-drying. A fim de garantir a estabilidade das enzimas durante um período de armazenamento, o extrato enzimático bruto (EEB) foi seco em spray-dryer sendo investigados os parâmetros operacionais e os adjuvantes de secagem para garantir a maior retenção da atividade enzimática após a secagem. Os ensaios foram divididos entre discriminação do adjuvante que proporcionasse maior proteção às enzimas, otimização das condições de secagem, armazenamento do EEB e dos pós secos, avaliação do efeito da secagem sobre as propriedades físico-químicas das endoglucanases e morfologia dos pós. Utilizou-se de planejamentos estatísticos para analisar os efeitos das variáveis temperatura de saída do ar de secagem, vazão de alimentação de solução, proporção entre o EEB e adjuvante, e teor de sólidos na formulação, sendo as respostas retenção de atividade enzimática e umidade dos pós. Nos ensaios de discriminação foi selecionado o adjuvante goma arábica. Em seguida, nos ensaios para otimização das condições de secagem, foi identificado menor vazão de alimentação e menores temperaturas de saída do ar de secagem para maiores retenções de atividade enzimática e maiores temperaturas e menores vazões para menores teores de umidade dos pós, o teor de sólidos revelou-se não significativo. O pó obtido nas condições mais favorável e menos favorável para a retenção da atividade enzimática foi armazenado à temperatura ambiente e sob refrigeração e a atividade enzimática determinada quinzenalmente. A análise estatística revelou não haver diferença na retenção da atividade enzimática após longos períodos para ambas as condições de secagem selecionadas e alternativas de armazenamento... / Abstract: This work aimed to provide technological treatment to enzymatic extract rich in endoglucanase, obtained by solid state culture using the fungus Myceliophtora thermophila M77, through spray-drying. Operational parameters and adjuvants were varied in order to obtain stable enzymes with high activities during storage. The experiments were divided into adjuvant discrimination, optimization of the drying conditions, storage of the raw enzymatic extract (REE) and powders, evaluation of drying on the physical-chemical characteristics of the endoglucanase, and powder morphology. Statistical experimental designs were used to evaluate the effect of the variables exit air temperature, solution flow rate, proportion REE/adjuvant, and total solid content on the enzyme activity retention and powder moisture content. The adjuvant Arabic gum was selected from the discrimination assays. From the optimization experiments, it was determined that the lower flow rate and air exit temperature resulted in the highest enzyme activity retention, while low temperature and solution flow rate resulted in the lowest retention. The total solid concentration was not statistically significant. The powders produced in the Best and in the worst drying conditions were stored at room temperature and under refrigeration and the enzymatic activity was measured at 15 days intervals. The statistical analysis did not show any difference in the enzymatic activity retention after long periods of storage for both drying conditions and both storage alternatives. The influence of temperature and pH on the enzyme activity was also investigated before and after the drying process, and it was noticed that these physical-chemical properties were not affected by the adjuvants and by the drying process. The morphology of the powders, obtained using the inert gas adsorption method, indicated that the powder obtained under the worst drying condition presented specific superficial area twice ... / Mestre

Thesis submitted in partial fulfilment of the requirements for the degree Doctor of Technology: Biomedical Technology In the Faculty of Health and Wellness Sciences At the Cape Peninsula University of Technology 2014 / Peroxidases are ubiquitous catalysts that oxidise a wide variety of organic and inorganic compounds employing peroxide as the electron acceptor. They are an important class of oxidative enzymes which are found in nature, where they perform diverse physiological functions. Apart from the white rot fungi, actinomycetes are the only other known source of extracellular peroxidases. In this study, the production of extracellular peroxidase in wild type actinomycete strains was investigated, for the purpose of large-scale production and finding suitable applications. The adjustment of environmental parameters (medium components, pH, temperature and inducers) to optimise extracellular peroxidase production in five different strains was carried out. Five Streptomyces strains isolated from various natural habitats were initially selected for optimisation of their peroxidase production. Streptomyces sp. strain BSII#1 and Streptomyces sp. strain GSIII#1 exhibited the highest peroxidase activities (1.30±0.04 U ml-1 and 0.757±0.01 U ml-1, respectively) in a complex production medium at 37°C and pH 8.0 in both cases. Maximum enzyme production for Streptomyces strain BSII#1 was obtained in the presence of 0.1 mM veratryl alcohol or pyrogallol, while 0.1 mM guaiacol induced the highest peroxidase production in Streptomyces sp. strain GSIII#1. As the highest peroxidase producer, Streptomyces sp. strain BSII#1 was selected for further studies. The strain was first characterised by a polyphasic approach, and was shown to belong to the genus Streptomyces using various chemotaxonomic, genotypic and phenotypic tests. Production of peroxidase was scaled up to larger volumes in different bioreactor formats. The airlift configuration was optimal for peroxidase production, with Streptomyces sp. strain BSII#1 achieving maximum production (4.76±0.46 U ml-1) in the 3 l culture volume within 60 hrs of incubation. A protocol for the purification of the peroxidase was developed, which involved sequential steps of acid and acetone precipitation, as well as ultrafiltration. A purification factor of at least 46-fold was achieved using this method and the protein was further analysed by LC-MS. The protein was shown to be a 46 kDa protein, and further biochemical characterisation showed that the peroxidase had a narrower spectrum of substrates as compared to reports on other peroxidases derived from actinomycetes. With 2,4-dichlorophenol as the substrate, the Km and Vmax for this enzyme were 0.893 mM and 1.081 μmol min-1, respectively. The purified peroxidase was also capable of catalysing coupling reactions between several phenolic monomer pairs. Overall, the peroxidase from Streptomyces sp. strain BSII#1 could feasibly be produced in larger scales and there remains further room to investigate other potential applications for this enzyme.

Orientador: Daniela Alonso Bocchini Martins / Coorientador: Eleni Gomes / Banca: Luis Henrique Souza Guimarães / Banca: João Cláudio Thoméo / Resumo: Devido à ampliação das aplicações industriais das celulases e hemicelulases, é crescente o interesse na produção destas enzimas por micro-organismos através de processos que promovam altos rendimentos a baixo custo. Assim, o cultivo de fungos filamentosos por fermentação em estado sólido (FES) utilizando-se resíduos lignocelulósicos como substratos tem sido frequentemente citado na literatura. Os fungos termofílicos tem se destacado neste sentido, por produzirem enzimas geralmente mais ativas e estáveis sob altas temperaturas, uma vantagem do ponto de vista das aplicações industriais. No presente trabalho, foram isolados 32 fungos termofílicos do ambiente a partir de amostras de silagem, compostagem e outros materiais orgânicos. Uma triagem inicial dos melhores produtores foi feita pelo cultivo dos isolados, por FES, em mistura de bagaço de cana-de-açúcar e farelo de trigo (1:1 p/p) como substratos, por 96h. A partir destes cultivos foram pré-selecionados 7 isolados, os quais foram então cultivados por FES em diferentes misturas de resíduos agro-industriais, por 96h, a fim de se avaliar a influência dos mesmos na produção das enzimas, buscando-se maiores rendimentos. Foram então selecionados para dar continuidade ao trabalho os isolados que mais se destacaram na produção das enzimas: Myceliophthora thermophila JCP 1-4 (endoglucanase, -glicosidase, xilanase e avicelase) e DABM 11/45 (-glicosidase, xilanase e avicelase). A influência do tempo de cultivo na produção das enzimas pelos dois isolados selecionados foi avaliada e as enzimas foram caracterizadas físico-quimicamente. O extrato enzimático produzido por M. thermophila, por FES, em mistura de bagaço de cana-de-açúcar e farelo de soja (1:1 p/p) foi utilizado para a sacarificação de bagaço de cana in natura e pré-tratado glicerol e explosão à vapor. Os bagaços in natura e pré-tratados foram... / Abstract: Due to the expansion of industrial applications of cellulases and hemicellulases, there is increasing interest in the production of these enzymes by micro-organisms through processes that promote high yields at low cost. Thus, the cultivation of filamentous fungi by solid state fermentation (SSF) using lignocellulose residues as substrates has been frequently cited in the literature. The thermophilic fungi has excelled in this regard, since they produce enzymes usually more active and stable under high temperatures, an advantage from the point of view of industrial applications. In this work, 32 thermophilic fungi were isolated from the environment, from samples of silage, composting and other organic materials. An initial screening of the best producers was made by cultivation of isolates, by SSF, in a mixture of sugar cane bagasse and wheat bran (1:1 w/w) as substrates, for 96h. From these cultivations 7 isolates were pre-selected, which were then cultured by SSF in different mixtures of agro-industrial residues, for 96h, in order to assess the influence of these substrates in enzymes production, seeking higher yields. Then, were selected to give continuity to the work the isolates that stood out in the production of enzymes: Myceliophthora thermophila JCP 1-4 (endoglucanase, -glucosidase, xylanase and avicelase) and DABM 11/45 (-glucosidase, xylanase and avicelase). The influence of the time on the enzymes production by the two selected isolates was assessed and the enzymes were characterized physico-chemically. The enzymatic extract produced by M. thermophila, by FES, in a mixture of sugar cane bagasse and soybean meal (1:1 w/w) was used for the saccharification of in natura and pre-treated glycerol and steam exploded sugarcane bagasse. The in natura and pre-treated bagasse were characterized regarding the content of cellulose, hemicellulose and lignin. The enzymatic extract produced by newly isolated fungus Myceliophtora... / Mestre

Neste trabalho, são descritos os detalhes da montagem de um sistema de cultivo microbiano em estado sólido, cujo elemento principal é um biorreator de tambor rotativo em escala de bancada, com casco em vidro refratário com aproximadamente 6,2 litros de volume. A montagem do equipamento exigiu estudos para a definição dos seguintes aspectos: geometria do reator; controle de frequência e período de agitação; umidificação, aquecimento e controle do fluxo do ar injetado no sistema; mistura do meio de cultivo; medição e controle da temperatura do meio; retirada de amostras. O sistema desenvolvido foi avaliado, tendo como processo fermentativo modelo o cultivo de Aspergillus niger T0005/007-2, microrganismo produtor de enzimas pectinolíticas. Os testes foram feitos em um meio contendo farelo de trigo como suporte sólido, avaliando-se a influência de três parâmetros principais agitação, massa de meio de cultivo e temperatura do meio sobre o crescimento celular e a produção de endo-poligalacturonase (PG) por A. niger. Três formas de agitação do meio sólido foram comparadas: sem agitação; agitação a 1 rpm por 5 minutos a cada duas horas; agitação a 1 rpm por 1 hora e 55 minutos a cada duas horas. A segunda forma de agitação levou ao melhor crescimento celular, 81 mg.g-1, e a atividade de endo-PG da ordem de 80 U.g-1, semelhante ao estimado com o sistema estático. Com a terceira forma de agitação, aparentemente houve dano ao micélio fúngico e, com isso, resultados inferiores foram alcançados. A avaliação do efeito da massa de meio de cultivo sobre o processo foi feita com cargas crescentes de meio que resultaram na ocupação de 30, 45 e 60 % do volume útil do reator. Quanto ao crescimento celular, o melhor resultado foi alcançado com a menor carga de meio, enquanto que a carga intermediária resultou no mais alto título enzimático final, 107,2 U.g-1. Nos experimentos sobre a influência da temperatura sobre o cultivo de A. niger, a maior atividade enzimática (80,6 U.g-1) foi obtida numa condição de trabalho que permitiu que a temperatura do meio atingisse valores da ordem de 45ºC. Com o meio mantido a 30ºC, a atividade enzimática máxima foi substancialmente mais baixa, 46,4 U.g-1. Nos testes realizados com o processo modelo, não se verificou uma clara influência positiva da agitação sobre a produção de endo-PG. Entretanto, a partir dos experimentos com temperatura controlada, é possível sugerir que a formação de endo-PG é favorecida por uma condição de estresse para o microrganismo, no caso representado por temperaturas do meio acima de 40ºC, visto que a temperatura ideal de crescimento de A. niger encontra-se na faixa de 28 a 34ºC. Este resultado, que discorda do que é genericamente descrito na literatura especializada sobre cultivos em estado sólido, é um exemplo da importância de dispor-se de um biorreator de tambor rotativo de pequena escala como equipamento básico para a realização de estudos fundamentais a respeito deste tipo de processo. Adicionalmente, o fato de o corpo do reator ser construído em vidro permite a visualização do espaço interno do tambor rotativo e observar o efeito da agitação sobre o meio de cultivo. / In this work, details on the assembling of a solid state cultivation system, whose main component is a bench scale rotating-drum bioreactor, are described. The bioreactor was built in refractory glass and has an approximate volume of 6.2 liters. To assemble the equipment, the following aspects have been studied: geometry of the bioreactor; control of frequency and period of agitation; moistening, warming and controlling of the inlet air flux; mixing of cultivation medium; determination and control of medium temperature; sample withdrawn. The developed system was evaluated, being the cultivation of the pectinolytic enzymeproducing microorganism Aspergillus niger T0005/007-2 used as the model process. The fermentative tests were carried out in a medium containing wheat-straw as solid support and were used to evaluate the effects of three main parameters agitation, mass of cultivation medium and medium temperature on the cell growth and the production of endopolygalacturonase (endo-PG) by A. niger. For agitation of the solid medium, three modes were compared: no agitation; agitation of 1 rpm for 5 minutes each 2 hours; agitation for 1 hour and 55 minutes, each 2 hours. The second mode of agitation led to the best cell growth, 81 mg.g-1, and to endo-PG activity of approximately 80 U.g-1, similar to that obtained with the static system. With the third mode of agitation occurred, apparently, some damage to fungus mycelium and inferior results were achieved. The evaluation of the mass of cultivation medium on the process was done by loading the reactor with masses that result in the occupation of 30, 45 and 60% of the working volume of the bioreactor. With respect to the cell growth, the best result was attained with the smallest load of medium, whereas the intermediate load resulted in the highest endo-PG activity, 107.2 U.g-1. In the experiments on the influence of the temperature on the cultivation of A. niger, the largest endo-PG activity (80.6 U.g-1) was obtained in a process condition that allowed that the temperature reached values close to 45ºC. When the medium temperature was controlled at 30ºC, the endo-PG activity was substantially lower, 46.4 U.g-1. In the tests with the model process, no clearly positive influence of agitation on the production of endo-PG was observed. From the temperature-controlled experiments however, it is possible to suggest that endo-PG formation is favored by a stress condition for the microorganism, represented in this case by medium temperatures over 40ºC, since the optimal temperatures for A. niger growth is found in the range of 28 to 34ºC. This result disagrees from what is generically described in the specialized literature for the solid state cultivations, being this fact an example of the importance of having a small-scale rotating-drum bioreactor as a basic equipment for fundamental studies on that type of fermentative process. Additionally, the fact of the reactor body being built on glass allows the inspection of the internal space of the rotating drum and to observe the effects of agitation on the cultivation medium.