The diploma thesis deals with the study of biotechnologically significant microorganisms, using the Raman spectroscopy. Content of the theoretical part is brief characteristic of Raman spectroscopy as a method, its use in practice and also use as a tool for monitoring of biotechnologically processes. Thesis was further focus on the biotechnologically significant microorganism Aureobasidium pullulans, its use in biotechnology and also for over-produced substances and in particular poly-L-maleic acid and pullulan. The content of the experimental part was study of selected strains A. pullulans, specifically stains as DSMZ, CCM F148 and CCM 8182, using Raman spectroscopy on the various types of culture media. Subject of practical part research was too production of extracellular polymers, acid poly-L-apple and pullulan, by selected strains A. pullulans. Objective of my thesis was described and determinate, spectra of individual strains as well as extracellular products, mainly pullulan, and then choose suitable production medium and optimal production strain A. pullulans. During experimental work was found, that optimal production strain was DSMZ strain culture on the mineral medium with the addition of yeast autolysate, which was optimal medium type. The content of the pullulan produced was for gravimetric determination, 6,3g/L, which also confirmed the results of the HPLC method. It was experimentally found, that Raman spectroscopy isn´t suitable method for quantification of extracellular products, but is appropriate and was used for PCA analysis of individual strains.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Xylanolytic accessory enzymes produced by the endo-p-l,4-xylanase overproducing, colour-variant strain of the euascomycetous fungus Aureobasidium pullulans, NRRL Y-2311-1, were studied. a- Glucuronidase activity was only induced during cultivation on carbon sources containing both xylose and glucuronic acid. An a-glucuronidase was partially purified from the supernatant of A. pullulans cultivated on birchwood glucuronoxylan. The enzyme had an apparent mobility on SDS-PAGE of 170 kDa, and after deglycosylation its mobility shifted to 118 kDa, indicating an extensively decorated protein. Maximal activity was measured at pH 3 in McIlvaine's phosphate-citrate buffer and at 40°C, and the enzyme was stable for 3 h at 40°C. The enzyme displayed substrate inhibition, and Km- and Kj-values were calculated as 3.3 ± 0.29 mM and 9.8 ± 3.8 mM for aldotriouronic acid and 29.5 ± 7.6 mM and 29.0 ± 7.8 for aldobiouronic acid respectively. PCR methods were used to clone the genes encoding an a-glucuronidase and an a-Larabinofuranosidase of A. pullulans NRRL Y-2311-1. The deduced amino acid sequence of the aglucuronidase encoding gene, aguA, shared greater than 60% identity with fungal glucuronidases and between 34% and 42% identity with bacterial a-glucuronidases, and it is member of family 67 of the glycoside hydrolases. The aguA gene encodes a protein of 836 amino acids with a putative secretion signal of 15 amino acids, resulting in a mature protein with a predicted molecular weight of 91 kDa. The gene was expressed in S. cerevisiae Y294 under control of the ADH2 promoter and terminator. The heterologous a-glucuronidase was purified to homogeneity using Ni-chelate affinity chromatography, and it had an electrophoretic mobility of 120 kDa on SDS-PAGE. The enzyme was maximally active at 65°C and between pH 5 and pH 6. The enzyme was stable at 45°C, lost half of its activity after 22.5 minutes at 55°C, and had a half-life of 5.6 min at 65 °C. It was stable at pH 4 and pH 6, and had a half-life of 17 min at pH 8. The enzyme had Km-values in the millimolar range for the series from aldobiouronic acid to aldopentaouronic acid. It had the highest catalytic efficiency on aldobiouronic acid and the catalytic efficiency decreased with increasing chain-length of the oligosaccharide substrate. The deduced amino acid sequence of the a-L-arabinofuranosidase gene, ab/A, shared between 69% and 76% identity with family 54 c-arabinofuranosidases. The gene encodes a polypeptide of 498 amino acids with a putative signal peptide of 20 amino acids resulting in a mature protein with a calculated molecular weight of 49.9 kDa. It was expressed in S. cerevisiae Y294 and the heterologous enzyme was purified to homogeneity by gel filtration. It's size estimated by gel filtration was 36 kDa, and it had an apparent mobility of 49 kDa on SDS-PAGE. It showed maximal activity at 55°C and between pH 3.5 and pH 4. It was stable at 50°C and between pH 4 and pH 5. The enzyme had a Km for p-nitrophenyl c-arabinofuranoside of 3.7 ± 0.36 mM and a Vrnax of 34.8 ± 1.1 U/mg protein. It displayed 0.2 U/mg activity against p-nitrophenyl ~-xylopyranoside. / AFRIKAANSE OPSOMMING: Hierdie studie het gefokus op xilanolitiese ensieme van die endo-I3-1,4-xilanase oorproduserende, kleur-variante ras van die euaskomiseet Aureobasidium pullulans, NRRL Y-2311-1. 0:- Glukuronidase-aktiwiteit is slegs geïnduseer tydens groei op koolstofbronne wat beide xilose en glukuronsuur bevat. u-Glukuronidase is gedeeltelik uit die supernatant van A. pullulans gekweek op berkehout glukuronoxilaan gesuiwer. Die ensiem se elekroforetiese mobiliteit met SDS-PAGE was 170 kDa en na deglikosilering het dit verskuif na 118 kDa, beduidend van 'n swaar geglikosileerde ensiem. Maksimum aktiwiteit is gemeet by pH 3 in McIlvaine se sitraat-fosfaat buffer en by 40°C. Die ensiem was stabiel by 40°C tydens 'n 3-uur inkubasie. Substraat inhibisie is bespeur, en die ensiem se Km- en Kj-waardes vir aldotriouronsuur was onderskeidelik 3.3 ± 0.29 mM en 9.8 ± 3.8 mM en vir aldobiouronsuur was die waardes onderskeidelik 29.5 ± 7.6 mM en 29.0 ± 7.8 mM. PKR metodes is benut om die gene vir u-glukuronidase en cc-arabinofuranosidase te kloneer. Die afgeleide aminosuurvolgorde van die c-glukuronidase geen, aguA, was meer as 60% identies aan swam cc-glukuronidases, en tussen 34% en 42% identies aan bakteriële u-glukuronidases, en dit is 'n lid van familie 67 van die glikosied hidrolases. Die aguA geen kodeer vir 'n proteïen van 836 amienosure met 'n sekresiesein van 15 amienosure, wat die produksie van 'n volwasse protein met 'n molekulêre gewig van 91 kDa tot gevolg het. Die geen is uitgedruk in S. cerevisiae Y294 onder beheer van die ADH2 promoter en termineerder. Ni-chelaat affiniteitschromatografie is gebruik om die heteroloë cc-glukuronidase te suiwer. Die elektroforetiese mobiliteit van die suiwer ensiem was 120 kDa met SDS-PAGE. Die ensiem het maksimale aktiwiteit by 65°C en tussen pH 5 en pH 6 getoon. Die ensiem was stabiel vir twee ure by 45°C, het die helfte van sy aktiwiteit binne 22.5 minute by 55°C verloor, en het 'n halfleeftyd van 5.6 minute by 65°C gehad. Dit was stabiel by pH 4 en pH 6 vir twee ure, en het 'n halfleeftyd van 17 minute by pH 8 gehad. Die ensiem het millimolaar Km-waardes getoon vir die substraatreeks vanaf aldobiouronsuur tot aldopentaouronsuur. Dit het die hoogste katalitiese effektiwiteit vir aldobiuronsuur gehad en die katalitiese effektiwiteit het afgeneem met toenemende lengte van die oligosakkaried substraat. Die afgeleide amienosuurvolgorde van die c-t-arabinofuranosidase geen, abfA, was tussen 69% en 76% identies aan familie 54 u-t-arabinofuranosidases. Die geen kodeer vir 'n proteïen van 498 amienosure met 'n seinpeptied van 20 aminosure, wat lei tot die produksie van 'n volwasse proteïen met 'n berekende molekulêre massa van 49.9 kDa. Die geen is uitgedruk in S. cerevisiae Y294 en die heteroloë ensiem is gesuiwer deur gel filtrasie. Die ensiem se geskatte molekulêre gewig met gel filtrasie was 36 kDa, en die ensiem se mobiliteit op SDS-PAGE was 49 kDa. Dit het maksimum aktiwiteit getoon by 55°C, en tussen pH 3.5 en pH 4. Dit was stabiel vir twee ure by 50°C en tussen pH 4 en pH 5. Die ensiem se Km vir p-nitrofeniel c-t-arabinofuranosied was 3.7 ± 0.36 mM en die Vmax was 34.8 ± 1.1 U/mg proteïen. Die ensiem het aktiwiteit teen p-nitrofenie1 I3-D-xilopiranosied van 0.2 U/mg getoon.

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-06-10T14:43:22Z No. of bitstreams: 1 Dissertação - Alessandra Alves Drumond Siqueira.pdf: 694552 bytes, checksum: 063f14c557446b37809da884d1b0d0a9 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-06-11T18:36:19Z (GMT) No. of bitstreams: 1 Dissertação - Alessandra Alves Drumond Siqueira.pdf: 694552 bytes, checksum: 063f14c557446b37809da884d1b0d0a9 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-06-11T18:39:17Z (GMT) No. of bitstreams: 1 Dissertação - Alessandra Alves Drumond Siqueira.pdf: 694552 bytes, checksum: 063f14c557446b37809da884d1b0d0a9 (MD5) / Made available in DSpace on 2015-06-11T18:39:17Z (GMT). No. of bitstreams: 1 Dissertação - Alessandra Alves Drumond Siqueira.pdf: 694552 bytes, checksum: 063f14c557446b37809da884d1b0d0a9 (MD5) Previous issue date: 2004-03-29 / Não Informada / The proteases are enzymes which occupy a pivotal position with respect to their application in medical and commercial fields. Collagenases are detached among several kind of proteases, used in pharmaceutical and cosmetician industry, in processes such as smooth the skin, oral hygiene, disinfect suppurative wounds, cicatrizing processes, burning and as a trombolytic agent. In order to select protease producing yeasts, this study had as objectives to partially characterize the enzyme as for pH and stability temperature and to verify the effect of the enzyme in the degradation of collagen. 50 samples of yeasts isolated from different substrates, obtained from the culture collection DPUA, were analysed. Samples were cultured in Malte extract supplemented with 1,0 % gelatin at 30 ºC in shaking conditions (140 rpm). The qualitative proteolytic activity was determined in agar gelatin-milk medium and the quantitative activity was determined using azocasein 1,0 % as substrate. The best culture conditions to protease production were established for a species, being studied the following parameters: characteristics of pathogenicity, size and age of inoculate, best growth temperature, coal and nitrogen sources. The best stability conditions of protease were assessed for pH (6-12) and temperature (25 º, 37 º, 40 º, 50 º, 60 º, 70 º and 80 ºC) and the effect of the enzyme in the degradation of collagen. Among the identified species, Trichosporon pullulans was selected, because it showed the highest levels of proteolytic activity in qualitative (halo = 27 mm) and quantitative (420 U/mL) assays. The assays to determine the characteristics of pathogenicity of this species showed positive growth at 37ºC, weak positive ureasic activity and there was no phospholipasic activity, the highest proteolytic activity (246,66 U/mL) detected with inoculate corresponded to 10% of the volume of medium and stock of culture growth for 24 hours. Trials to verify the effect of temperature (25 ºC to 50 ºC) on growth and protease production demonstrated that the higher biomass (3,46 mg/mL) and proteolytic activity (513,33 U/mL) were obtained at 30 ºC. Sucrose 1,0 % was the best coal source yielding higher biomass (6,18 mg/mL) and proteasic activity (580 U/mL) and as nitrogen source detached peptone 0,5 % showing the higher values of proteolytic activity (755,55 U/mL) and biomass (7,55 mg/mL). The collagenolytic activity was determined in solid medium using dissolvable collagen as the only coal source. T. pullulans degraded collagen, producing a 28 mm halo. This work demonstrated that protease, with collagenolytic activity; produced by T. pullulans, can be used as therapeutic agent further detailed studies. / As proteases são enzimas que ocupam uma posição central com respeito à aplicação na área médico e industrial. Entre os vários tipos de proteases, utilizadas na indústria farmacêutica e de cosméticos, destacam-se as colagenases utilizadas para amaciamento da pele, higiene oral, na limpeza de feridas purulentas, em processos de cicatrização, queimaduras e como agente anti-trombolítico. Realizou-se este estudo com o objetivo de selecionar leveduras produtoras de proteases, avaliar as melhores condições de crescimento da espécie produtora de protease, caracterizar parcialmente a enzima quanto o pH e temperatura na estabilidade e verificar o efeito desta enzima na degradação do colágeno. Foram analisadas 50 amostras de leveduras isoladas de diferentes substratos, provenientes da coleção de cultura DPUA. As amostras foram cultivadas em extrato de Malte suplementado com gelatina 1,0 % a 30 ºC, sob agitação (140 rpm) por 72 horas. A atividade proteolítica qualitativa foi determinada em meio sólido ágar gelatina-leite e a quantitativa utilizando como substrato azocaseína 1 % p/v. Foram estabelecidas, para a espécie selecionada, as melhores condições de cultivo para produção da protease, foram estudados: características de patogenicidade, tamanho e idade do inóculo, temperatura ótima de crescimento, fontes de carbono e nitrogênio. Foram avaliadas as melhores condições de estabilidade da protease pH (6-12) e temperatura (25 º, 37 º, 40 º, 50 º, 60 º, 70 º e 80 ºC) e também, o efeito da enzima na degradação do colágeno. A determinação da atividade colagenolítica foi realizada em meio sólido utilizando colágeno solúvel como única fonte de carbono. Selecionou-se, entre as espécies identificadas, Trichosporon pullulans (DPUA), por apresentar o maior valor de atividade proteolítica em ensaio qualitativo (halo = 27 mm) e em ensaio quantitativo (420 U/mL). Os experimentos realizados para detectar as características de patogenicidade desta espécie demonstraram crescimento positivo a 37 ºC, atividade ureásica positiva e não foi detectada atividade fosfolipásica; a maior atividade proteolítica (246,66 U/mL) foi detectada com inóculo que correspondeu a 10 % do volume de meio e cultura estoque de 24 horas de crescimento. Os ensaios realizados para verificar o efeito da temperatura no crescimento e na produção da protease demonstraram que a maior biomassa (3,46 mg/mL) e atividade proteolítica (513,33 U/mL) foram obtidas a 30 ºC. A melhor fonte de carbono foi sacarose 1,0 % produzindo maior crescimento (6,18 mg/mL) e maior atividade proteásica (580 U/mL) e como fonte de nitrogênio destacou-se a peptona 0,5 % expressando os maiores valores de atividade proteolítica (755,55 U/mL) e biomassa de 7,55 mg/mL. Os resultados obtidos neste estudo sugerem que a protease produzida por T. pullulans classifica-se como protease alcalina (pH ótimo 8,0). E maior estabilidade térmica da protease foi observada a 37 ºC onde a enzima manteve 100 % de sua atividade durante 40 minutos de incubação. T. pullulans degradou o colágeno produzindo halo de 28 mm. Esse estudo demonstrou que a protease apresenta atividade colagenolítica e poderá ser utilizada como agente terapêutico

Biossurfactantes são moléculas anfifílicas produzidas por micro-organismos que possuem grande potencial na substituição de surfactantes químicos, pois apresentam maior biodegradabilidade e estabilidade. A busca por novos micro-organismos, matérias-primas e estratégias de produção é essencial para a viabilização da sua produção. Assim, a fermentação em estado sólido (FES) apresenta-se como uma tecnologia alternativa de produção, com a vantagem de evitar a formação de espuma, e a utilização de leveduras para o processo é vantajosa, pois muitas não apresentam risco de patogenicidade. O objetivo deste trabalho foi selecionar uma levedura capaz de produzir biossurfactante por FES, determinar as condições do processo, comparar com a fermentação submersa (FS), caracterizar bioquimicamente o biossurfactante e testar sua aplicação para biorremediação. Para tanto, 37 leveduras foram avaliadas quanto à produção de biossurfactante em caldo Kitamoto, por meio de testes das atividades tensoativa e emulsificante. Dessas, 17 apresentaram resultados positivos para tensoatividade, formaram emulsão estável e foram utilizadas para os testes em FES em 2 g bagaço de cana-de-açúcar e 10 mL de meio Kitamoto. Na FES, cinco leveduras apresentaram resultados positivos para tensoatividade e quatro formaram emulsão estável. Dentre essas, a Aureobasidium pullulans LB 83 foi selecionada por apresentar resultado positivo para tensoatividade, índice de emulsificação acima de 50% e estabilidade da emulsão. Foram testadas diferentes fontes de carbono, sendo a sacarose aquela que apresentou melhores resultados (6,0 cm de tensoatividade no teste de espalhamento da gota (Ta) e 8,3x10-2 cm/h de produtividade em tensoatividade (QTa)). A adição dos indutores glicerol (0 a 6 g/L) e óleo de soja (0 a 10 g/L) não apresentou efeito significativo para o processo. Também foi estudada a influência da aeração (0,1 a 1,1 h-1) e da concentração de sacarose (20 a 80 g/L) utilizando planejamento fatorial composto de face centrada realizado em reator de tanque agitado. O uso das variáveis no nível mais alto aumentou a produção de biossurfactante (8,05 cm (Ta) e 8,4x10-2 cm/h (QTa). As condições adequadas da FES foram avaliadas em frascos Erlenmeyer (50 mL) com 2 g de bagaço (suporte inerte) em um planejamento 24, tendo como variáveis tamanho médio das partículas de bagaço (0,6 a 1,8 mm), volume de meio adicionado (8 a 12 mL), concentração celular inicial (1x105 a 1x107 cel/mL) e volume para extração (15 a 25 mL). O tamanho das partículas apresentou efeito positivo e o volume de meio possuiu efeito negativo na concentração de biossurfactante. As demais variáveis não apresentaram efeitos significativos. Assim, as condições definidas foram 1,18 mm tamanho médio de partícula, 1x106 cel/mL concentração celular, 8 mL meio de cultura e 15 mL volume de extração, resultando na obtenção de 2,06 g/L de biossurfactante. Não houve diferença significativa entre o rendimento da condição otimizada na FES e a FS. A utilização de butanona para a extração mostrou-se vantajosa e o biossurfactante foi caracterizado como poliol lipídeo. Sua aplicação para biorremediação foi avaliada e apresentou maiores recuperações de petróleo da areia contaminada (73,7 e 78,4%) que as obtidas por dodecil sulfato sódico (58,0 e 75,0%), nas concentrações de 0,1 e 0,5 % respectivamente. Concluiu-se que a levedura A. pullulans LB 83 foi capaz de produzir biossurfactante por FES e esse processo apresenta destacada potencialidade, podendo servir como conhecimento para futuros estudos visando sua implementação em escalas maiores. / Biosurfactants are amphiphilic molecules produced by microorganisms that have great potential as substitute for chemical surfactants, since they present higher biodegradability and stability. The search for new microorganisms, raw materials and production strategies are essential for their production viability. Thus, solid state fermentation (FES) is presented as an alternative production technology, with the advantage of no foam formation, and the selection of yeasts for the process is favorable, since many of them do not present risk of pathogenicity. The objective of this work was to select a yeast able to produce biosurfactant by FES, to determine process conditions, to compare the results obtained by FES with the process of submerged fermentation (FS), to characterize biochemically the biosurfactant and to test its application for bioremediation. For this, 37 yeasts were evaluated for biosurfactant production in Kitamoto broth, using tests of tensoactive and emulsifying activities. 17 presented positive results for tensoativity and were able to form stable emulsion, and were used in tests of FES using 2 g of sugarcane bagasse and 10 mL of Kitamoto medium. In FES, five yeasts presented positive results for tensoativity and four were able to form a stable emulsion. Among these, Aureobasidium pullulans LB 83 was selected due to its positive results for tensoativity, emulsification index above 50% and emulsion stability. Different carbon sources were tested for biosurfactant production by A. pullulans LB 83 and sucrose presented the best results (6.0 cm of tensoativity in drop spreading test (Ta) and 8.3x10-2 cm/h of tensoactivity productivity (QTa). The addition of the inductors glycerol (0 to 6 g/L) and soybean oil (0 to 10 g/L) had no significant effect on the biosurfactant production process. The influence of aeration (0.1 to 1.1 h-1) and sucrose concentration (20 to 80 g/L) were also studied using factorial composite face centered design in a stirred tank reactor. The use of the variables at the highest level increased biosurfactant production (8.05 cm (Ta) and 8.4 x 10-2 cm/h (QTa). The appropriate conditions for FES process were evaluated in Erlemeyers flasks (50 mL) with 2 g of sugarcane bagasse (inert support) in a factorial design 24. The variable used were bagasse particles size (0.6 to 1.8 mm), medium volume added (8 to 12 mL), initial cell concentration (1x105 to 1x107 cell/mL) and water volume for extraction (15 to 25 mL). Particle size had a positive effect and medium volume had a negative effect on biosurfactant concentration. The other variables did not present significant effects. Thus, the defined conditions were 1.18 mm of particle size, 1x106 cell/mL of initial cell concentration, addition of 8 mL of culture medium and 15 mL for extraction volume (2.06 g/L of biosurfactant was obtained). There was no significant difference between the performance of the optimized condition in FES and FS. The use of butanone for the extraction was advantageous and the biosurfactant was characterized as polyol lipid. Its application for bioremediation was evaluated, exhibiting a higher recovery of contaminated sand oil (73.7 and 78.4%) than those obtained by sodium dodecyl sulphate (58.0 and 75.0%), at concentrations of 0.1 and 0.5% respectively. For these results, it was concluded that the yeast A. pullulans LB 83 was able to produce biosurfactant by FES and this process has outstanding potential, and can be used for future studies aimed at implementation of larger scales.

Biossurfactantes são moléculas anfifílicas produzidas por micro-organismos que possuem grande potencial na substituição de surfactantes químicos, pois apresentam maior biodegradabilidade e estabilidade. A busca por novos micro-organismos, matérias-primas e estratégias de produção é essencial para a viabilização da sua produção. Assim, a fermentação em estado sólido (FES) apresenta-se como uma tecnologia alternativa de produção, com a vantagem de evitar a formação de espuma, e a utilização de leveduras para o processo é vantajosa, pois muitas não apresentam risco de patogenicidade. O objetivo deste trabalho foi selecionar uma levedura capaz de produzir biossurfactante por FES, determinar as condições do processo, comparar com a fermentação submersa (FS), caracterizar bioquimicamente o biossurfactante e testar sua aplicação para biorremediação. Para tanto, 37 leveduras foram avaliadas quanto à produção de biossurfactante em caldo Kitamoto, por meio de testes das atividades tensoativa e emulsificante. Dessas, 17 apresentaram resultados positivos para tensoatividade, formaram emulsão estável e foram utilizadas para os testes em FES em 2 g bagaço de cana-de-açúcar e 10 mL de meio Kitamoto. Na FES, cinco leveduras apresentaram resultados positivos para tensoatividade e quatro formaram emulsão estável. Dentre essas, a Aureobasidium pullulans LB 83 foi selecionada por apresentar resultado positivo para tensoatividade, índice de emulsificação acima de 50% e estabilidade da emulsão. Foram testadas diferentes fontes de carbono, sendo a sacarose aquela que apresentou melhores resultados (6,0 cm de tensoatividade no teste de espalhamento da gota (Ta) e 8,3x10-2 cm/h de produtividade em tensoatividade (QTa)). A adição dos indutores glicerol (0 a 6 g/L) e óleo de soja (0 a 10 g/L) não apresentou efeito significativo para o processo. Também foi estudada a influência da aeração (0,1 a 1,1 h-1) e da concentração de sacarose (20 a 80 g/L) utilizando planejamento fatorial composto de face centrada realizado em reator de tanque agitado. O uso das variáveis no nível mais alto aumentou a produção de biossurfactante (8,05 cm (Ta) e 8,4x10-2 cm/h (QTa). As condições adequadas da FES foram avaliadas em frascos Erlenmeyer (50 mL) com 2 g de bagaço (suporte inerte) em um planejamento 24, tendo como variáveis tamanho médio das partículas de bagaço (0,6 a 1,8 mm), volume de meio adicionado (8 a 12 mL), concentração celular inicial (1x105 a 1x107 cel/mL) e volume para extração (15 a 25 mL). O tamanho das partículas apresentou efeito positivo e o volume de meio possuiu efeito negativo na concentração de biossurfactante. As demais variáveis não apresentaram efeitos significativos. Assim, as condições definidas foram 1,18 mm tamanho médio de partícula, 1x106 cel/mL concentração celular, 8 mL meio de cultura e 15 mL volume de extração, resultando na obtenção de 2,06 g/L de biossurfactante. Não houve diferença significativa entre o rendimento da condição otimizada na FES e a FS. A utilização de butanona para a extração mostrou-se vantajosa e o biossurfactante foi caracterizado como poliol lipídeo. Sua aplicação para biorremediação foi avaliada e apresentou maiores recuperações de petróleo da areia contaminada (73,7 e 78,4%) que as obtidas por dodecil sulfato sódico (58,0 e 75,0%), nas concentrações de 0,1 e 0,5 % respectivamente. Concluiu-se que a levedura A. pullulans LB 83 foi capaz de produzir biossurfactante por FES e esse processo apresenta destacada potencialidade, podendo servir como conhecimento para futuros estudos visando sua implementação em escalas maiores. / Biosurfactants are amphiphilic molecules produced by microorganisms that have great potential as substitute for chemical surfactants, since they present higher biodegradability and stability. The search for new microorganisms, raw materials and production strategies are essential for their production viability. Thus, solid state fermentation (FES) is presented as an alternative production technology, with the advantage of no foam formation, and the selection of yeasts for the process is favorable, since many of them do not present risk of pathogenicity. The objective of this work was to select a yeast able to produce biosurfactant by FES, to determine process conditions, to compare the results obtained by FES with the process of submerged fermentation (FS), to characterize biochemically the biosurfactant and to test its application for bioremediation. For this, 37 yeasts were evaluated for biosurfactant production in Kitamoto broth, using tests of tensoactive and emulsifying activities. 17 presented positive results for tensoativity and were able to form stable emulsion, and were used in tests of FES using 2 g of sugarcane bagasse and 10 mL of Kitamoto medium. In FES, five yeasts presented positive results for tensoativity and four were able to form a stable emulsion. Among these, Aureobasidium pullulans LB 83 was selected due to its positive results for tensoativity, emulsification index above 50% and emulsion stability. Different carbon sources were tested for biosurfactant production by A. pullulans LB 83 and sucrose presented the best results (6.0 cm of tensoativity in drop spreading test (Ta) and 8.3x10-2 cm/h of tensoactivity productivity (QTa). The addition of the inductors glycerol (0 to 6 g/L) and soybean oil (0 to 10 g/L) had no significant effect on the biosurfactant production process. The influence of aeration (0.1 to 1.1 h-1) and sucrose concentration (20 to 80 g/L) were also studied using factorial composite face centered design in a stirred tank reactor. The use of the variables at the highest level increased biosurfactant production (8.05 cm (Ta) and 8.4 x 10-2 cm/h (QTa). The appropriate conditions for FES process were evaluated in Erlemeyers flasks (50 mL) with 2 g of sugarcane bagasse (inert support) in a factorial design 24. The variable used were bagasse particles size (0.6 to 1.8 mm), medium volume added (8 to 12 mL), initial cell concentration (1x105 to 1x107 cell/mL) and water volume for extraction (15 to 25 mL). Particle size had a positive effect and medium volume had a negative effect on biosurfactant concentration. The other variables did not present significant effects. Thus, the defined conditions were 1.18 mm of particle size, 1x106 cell/mL of initial cell concentration, addition of 8 mL of culture medium and 15 mL for extraction volume (2.06 g/L of biosurfactant was obtained). There was no significant difference between the performance of the optimized condition in FES and FS. The use of butanone for the extraction was advantageous and the biosurfactant was characterized as polyol lipid. Its application for bioremediation was evaluated, exhibiting a higher recovery of contaminated sand oil (73.7 and 78.4%) than those obtained by sodium dodecyl sulphate (58.0 and 75.0%), at concentrations of 0.1 and 0.5% respectively. For these results, it was concluded that the yeast A. pullulans LB 83 was able to produce biosurfactant by FES and this process has outstanding potential, and can be used for future studies aimed at implementation of larger scales.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: The euascomycetous fungus Aureobasidium pullulans produces xylanolytic accessory enzymes, including an α-L-arabinofuranosidase. The deduced amino acid sequence of the abfA gene encoding α-L-arabinofuranosidase was 69-76% identical to family 54 glycoside hydrolases. The abfA gene encoded a 498 amino acid polypeptide including a signal peptide consisting of 20 amino acids. The mature protein had a calculated molecular weight of 49.9 kDa. One putative N-glycosylation site was found and an iso-electric point of 4.97 was calculated. A. pullulans AbfA was also found to consist of an N-terminal catalytic domain (residues 1-317) and a C-terminal arabinose-binding domain (residues 318-442). The abfA gene from the colour-variant strain of A. pullulans, NRRL Y-2311-1, was recently transferred in Saccharomyces cerevisiae Y294. The yeast culture was grown on synthetic defined medium and α-L-arabinofuranosidase was expressed successfully, secreted from the cells and was purified from the supernatant in a single step using gel filtration. It had an apparent mobility of 52.7 kDa on SDS-PAGE and 36 kDa estimated by gel filtration. The heterologous enzyme was characterized according to pH and temperature dependence and stability, apparent mobility and kinetic properties. The temperature optimum of the recombinant α–L-arabinofuranosidase was 55 °C and it was stable over 3 h at 40 °C. The enzyme displayed optimum activity between pH 4 and 4.5 and was stable at pH 4 over 3 h. Kinetic analysis on p-nitrophenyl-α-arabinofuranoside yielded a Km of 1.43 mM and a Vmax of 23.7 U/mg. Product inhibition was observed and a Ki of 28 ± 3 mM was determined during assaying in the presence of arabinose. A specific activity of 3.85 ± 0.008 U/mg was determined on p-nitrophenyl-α-L-arabinofuranoside and no activity was found on chromogenic substrates which contained a β-linked arabinofuranosyl. The enzyme showed low activity against the 1,5-α-L-arabino-oligosaccharides and cleaved arabinose from corn fibre, oat spelt arabinoxylan and to a lesser degree wheat arabinoxylan. No release of arabinose was observed from larch wood arabinogalactan, α-1,5-debranched arabinan and lignin-arabinose substrates. Linkage preference showed less activity against α-1,5-linked than α-1,2 or α-1,3-linked arabinofuranosyl subunits. Synergism between α–L-arabinofuranosidase and endo-β-1,4-xylanase occurred when measuring the increase in arabinose during wheat arabinoxylan degradation. A. pullulans NRRL Y-2311-1 was grown on synthetic defined medium and native α-L-arabinofuranosidase was expressed and secreted into the culture medium. The native enzyme was partially purified from the supernatant in two steps using gel filtration. The native α–L-arabinofuranosidase had an apparent mobility of 51.5 kDa on SDS-PAGE, displayed optimum activity at 50°C and pH 3. Kinetic analysis on p-nitrophenyl-α-arabinofuranoside gave a Km of 8.33 mM and a Vmax of 1.54 U/mg, and the enzyme showed slight activity against 1,5-α-L-arabinotriose. The properties of the native enzyme were similar to that of the heterologous α–L-arabinofuranosidase. Hydrolysis of sugar cane bagasse by heterologous α–L-arabinofuranosidase and xylanase revealed that pre-treatment with liquid ammonium was more effective in releasing component sugars than a pre-treatment with water at 140º C. A three-dimensional homology model of the heterologous α–L-arabinofuranosidase was constructed using the solved crystal structure of arabinofuranosidase (AkabfB) from Aspergillus kawachii, which was 71 % identical. / AFRIKAANSE OPSOMMING: Die euaskomisetiese swam Aureobasidium pullulans produseer xilanolitiese ensieme, insluitend ‘n α-L-arabinofuranosidase. Die afgeleide aminosuurvolgorde van die abfA geen wat α-L-arabinofuranosidase enkodeer, was 69-76% identies aan familie 54 glikosied hidrolase. Die abfA geen enkodeer ‘n 498 aminosure polipeptied, insluitend ‘n sein peptied bestaande uit 20 aminosure. Die volwasse proteïen het ‘n berekende molekulêre gewig van 49.9 kDa. Een moontlike N-glikosilasie plek is gevind en ‘n iso-elektriese punt van 4.97 is bereken. A. pullulans AbfA bestaan uit ‘n N-terminaal katalitiese gebied (residu 1-317) en ‘n C-terminaal arabinose-bindingsgebied (residu 318-442). Die abfA geen van die kleur-variante ras van A. pullulans, NRRL Y-2311-1, is onlangs na Saccharomyces cerevisiae Y294 oorgedra. Die giskultuur is op ‘n sinteties gedefinieërde medium gegroei en α-L-arabinofuranosidase was suksesvol uitgedruk, uitgedra van af die selle en van uit die supernatant gesuiwer in ‘n enkele stap deur gel filtrasie te gebruik. Dit het ‘n berekende mobiliteit van 52.7 kDa op SDS-PAGE en 36 kDa geskat deur middel van gel filtrasie. Die heteroloë ensiem is gekarakteriseer volgens pH en temperatuurafhanklikheid en stabiliteit, klaarblyklike mobiliteit en kinetiese eienskappe. Die temperatuur optimale van α–L-arabinofuranosidase was 55 °C en dit was stabiel oor 3 ure teen 40 °C. Die ensiem het optimale aktiwiteit tussen pH 4 en 4.5 getoon en was stabiel teen pH4 en oor 3 ure. Kinetiese analiese op p-nitrofeniel-α-L-arabinofuranosied het ‘n Km van 1.43 gelewer en ‘n Vmax van 23.7 U/mg. Produkinhibisie is opgelet en ‘n Ki van 28 ± 3 mM is vasgestel gedurende toetsing in die teenwoordigheid van arabinose. ‘n Spesifieke aktiwiteit van 3.85 ± 0.008 U/mg is vasgestel op p-nitrofeniel-α-L-arabinofuranosied en geen aktiwiteit was gevind op chromogeniese substrate wat ‘n β-verbinding arabinofuranosiel bevat het nie. Die ensiem het ‘n lae aktiwiteit getoon teen die 1,5-α-L-arabino-oligosakkaried en het die arabinose van mielievesel, hawerspelt arabinoxilaan en tot ‘n mindere mate koring arabinoxilaan geskei. Geen vrystelling van arabinose is van af lorkehout arabinogalactan, α-1,5-onvertakte arabinan en lignin-arabinose substrate nie opgemerk. Verbindingsvoorkeure het minder aktiwiteit teen α-1,5-verbindings as α-1,2 of α-1,3- verbindings arabinofuranosiel subeenhede getoon. Sinergisme tussen α–L-arabinofuranosidase en endo-β-1,4-xilanase het plaasgevind met die bepaling van meer arabinose gedurende koring arabinoxilaan degradasie. A. pullulans NRRL Y-2311-1 is op sinteties gedefinieerde medium gegroei en α-L-arabinofuranosidase was uitgedruk en uitgeskei in die kultuur medium. Die inheemse ensiem was gedeeltelik gesuiwer van die supernatant in twee stappe met die gebruik van gel filtrasie. Die inheemse α–L-arabinofuranosidase het ‘n klaarblyklike mobiliteit van 51.5 kDa op SDS-PAGE, het optimum aktiwiteit vertoon by 50°C en pH 3. Kinetiese analiese op p-nitrofeniel-α-arabinofuranosiede het ‘n Km van 8.33 mM en ‘n Vmax van 1.54 U/mg, en die ensiem het effense aktiwiteit teen 1,5-α-L-arabinotrios getoon. Die eienskappe van die inheemse ensiem was soortgelyk aan die van die heteroloë α–L-arabinofuranosidase. Hidroliese van suikerrietbagasse met heteroloë α–L-arabinofuranosidase en xilanase het aan die lig gebring dat vooraf behandeling met vloeistof ammonium meer effektief is in die vrystelling van komponent suikers as ‘n vooraf behandeling met water teen 140º C. ‘n Driedimensionele homologiese model van die heteroloë α–L-arabinofuranosidase is gekonstrueer deur die gebruik van die verklaarde kristal struktuur van arabinofuranosidase (AkabfB) van Aspergillus kawachii, wat 71% identies was.

Made available in DSpace on 2014-06-11T19:31:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-05Bitstream added on 2014-06-13T19:19:58Z : No. of bitstreams: 1 tobal_tm_dr_arafcf.pdf: 1024748 bytes, checksum: 9a431e076fe886b2b80c19c23adb8bde (MD5) / Universidade Estadual Paulista (UNESP) / Este estudo teve como objetivo aplicar β-glicosidases produzidas por microrganismos taxonomicamente distantes do gênero Saccharomyces na produção de aguardente de cana, visando uma maior liberação de compostos flavorizantes, em especial os monoterpenos para a obtenção de aguardentes com atributo sensorial floral mais intenso. Testes preliminares foram realizados adicionando-se extrato bruto de β-glicosidases do Aureobasidium pullulans ER-16 e do Thermoascus aurantiacus CBMAI-756 no caldo de quatro variedades de cana-de-açúcar, com pH 4.5 e diferentes condições de temperatura, agitação e tempo de hidrólise. O complexo enzimático produzido pelo microrganismo T. aurantiacus foi adicionado anteriormente à inoculação do caldo de cana com a Saccharomyces cerevisiae, e a fermentação padrão com S. cerevisiae foi adotada como referência. A determinação dos efeitos das β-glicosidases na desglicosilação de terpenos foi avaliada utilizando um cromatógrafo gasoso acoplado a um espectrômetro de massas (GC-MS), com injeção da fase vapor do mosto e dos produtos de destilação no modo head-space com micro extração em fase sólida (HS-SPME), além da aplicação de testes de análise sensorial. Nenhum composto terpênico foi encontrado nos caldos originais, entretanto citronelol, nerol e geraniol foram encontrados nos caldos de cana tratados com β-glicosidases, na faixa de microgramas por litro, exceto para variedade IAC87-3396. Temperatura, tempo de hidrólise e agitação não afetou os valores encontrados, entretanto uma maior eficiência da atividade hidrolítica de β-glicosidases do T. aurantiacus foi encontrada. Não foi observada a presença de linalol e nerol na cahaça controle até o limite de detecção de 5 μg/L, entretanto a presença de linalol e nerol foi confirmada na cachaça tratada... / This research aims to apply β-glucosidase produced by microorganisms taxonomically distant from the genus Saccharomyces to produce Brazilian sugar cane spirit, seeking a greater release of flavor compounds, especially monoterpenes to brandies with obtaining sensory attribute floral more intense. The enzymatic complexe by Aureobasidium pullulans ER-16 and Thermoascus aurantiacus CBMAI-756 were added to the juices from four sugar cane varieties, with pH 4.5 and under different conditions of temperature, agitation and hydrolysis time. The enzyme complexe produced by microorganism Thermoascus aurantiacus, was added before the inoculation of sugarcane juice with Saccharomyces cerevisiae and the fermentation pattern of S. cerevisiae was used as a reference. The determination of the effects of the addition of β-glucosidases in the deglycosylation of terpenes was evaluated by gas chromatography with mass spectrometry (GC-MS), with injection of the vapor of the distillates in order to head-space micro-phase extraction solid (HS-SPME), and sensory evaluation. No terpenic compounds were found in the original juices, but citronellol, nerol and geraniol were found in the treated sugar cane juices in the range of micrograms per liter, except for variety IAC87-3396. Temperature, hydrolysis time and agitation did not show to be significant parameters in this process, however a greater efficiency of the hydrolytic activity of β-glucosidases from the T. aurantiacus was found. Linallol and nerol weren`t detected in the control cachaça at LOD of 5 μg/L, whereas in the enzymatic treated cachaça, the presence of linallol and nerol was confirmed. The concentrations of terpienol and geraniol were significantly increased in treated sugar cane juice spirit, which received higher scores in the sensory evaluation.

Orientador: Eleni Gomes / Resumo: O grande acumulo de biomassa lignocelulósica oriunda da produção de etanol no Brasil contribui para um aumento em pesquisas para utilização para etanol de segunda geração e produção de enzimas com maior valor agregado. Considerando que o grupo de pesquisa ao qual esse trabalho está vinculado tem desenvolvido pesquisas com celulases, hemicelulases, ligninases, pectinases, esse estudo teve por objetivo selecionar leveduras produtoras dessas enzimas, realizar a caracterização físico-química e a purificação enzimática. Diversos trabalhos tem sido desenvolvidos a fim do reaproveitamento de material lignocelulósico oriundo da produção de etanol por cana-de-açúcar para a produção de enzimas de interesse comercial e sua principal aplicação na produção de etanol de segunda geração. Foi selecionado duas leveduras entre 30 escolhidas da coleção do Laboratório de Bioquímica e Microbiologia Aplicadas UNESP/IBILCE: A. pullulans LB 3.1 e A. leucospermi LB 86 e que foram cultivadas com farelo de trigo. Ambas foram capazes de produzir xilanase, β-xilosidase e β-glicosidase, sendo a maior atividade de xilanase de 72 U ml-1 para LB 3.1 de 5,5 U ml-1 para LB 86, bem como a atividade de β-xilosidase que foi (6,7 U ml-1 ) para LB 3.1 e (4,0 U ml-1 ). Com relação à influência do pH na atividade enzimática foi observado perfis similares para xilanase e βxilosidase produzidas pela levedura A. pullulans LB 3.1, com uma maior atividade na faixa de pH 2,5 a 3,5. Essa característica foi observada também ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The large accumulation of lignocellulosic biomass from ethanol production in Brazil contributes to an increase in research for use for second generation ethanol and the production of enzymes with greater added value. Considering that the research group to which this work is linked has developed research with cellulases, hemicellulases, ligninases, pectinases, this study aimed to select yeasts that produce these enzymes, perform physical-chemical characterization and enzymatic purification. Several works have been developed in order to reuse lignocellulosic material from the production of ethanol by sugarcane for the production of enzymes of commercial interest and its main application in the production of second generation ethanol. Two yeasts were selected from 30 chosen from the collection of the Laboratory of Applied Biochemistry and Microbiology UNESP / IBILCE: A. pullulans LB 3.1 and A. leucospermi LB 86 and which were grown with wheat bran. Both were able to produce xylanase, β-xylosidase and β-glycosidase, with the highest xylanase activity being 72 U ml-1 for LB 3.1 and 5.5 U ml-1 for LB 86, as well as the activity of β-xylidasidase which was (6.7 U ml-1) for LB 3.1 and (4.0 U ml-1). Regarding the influence of pH on enzyme activity, similar profiles were observed for xylanase and β-xylosidase produced by the yeast A. pullulans LB 3.1, with a greater activity in the pH range 2.5 to 3.5. This characteristic was also observed for the βxylosidase of A. leucospermi LB 86, w... (Complete abstract click electronic access below) / Doutor

The waste from agricultural and food industry are accessible in large quantity anywhere in the whole world nowadays. Most of these wastes include cellulose (30 - 40 %), hemicellulose (20 - 40 %) and lignine (10 - 20 %). Therefore these waste materials have wide use as the substrates for the microbial growth and the production of the enzymes. The microorganisms are able to use organic compounds from the wastes as the source of energy for the growth and carbon for synthesis of cellular biomass [24]. Wheat and rice straw are possible to use as the substrates for cultivation of the microorganisms and following production of the enzymes. In this thesis the utilization of the wastes from food industry for the production of the enzymes by the microorganisms was studied. We observed utilization of wheat straw as source of energy for growth of tested microorganisms and investigated their ability for the production of oxidoreductase (laccase). The optimalization of growth conditions of Aureobasidium pullulans was proceeded. Further the activity of laccase was studied. Milled wheat straw was used as the substrate. The cultivation was done in the thermoregulator at the temperature of 27°C. The activity of laccase was not found in this thesis. Petri dishes were contaminated by three unknown microoganisms during optimalization of growth of Aureobasidium pullulans. One of them produced laccase in cultivation with straw.

The study of production of hydrolytic enzymes dealt with the production of cellulase and polygalacturonase by two microbial strains - Aspergillus niger and Aureobasidium pullulans. The enzymes were produced in solid-state fermentation system. The wheat straw and sugar beet pulp were used as a substrate. The substrates were moistened by water, mineral solution or by medium with glucose. The effect of mineral solution and glucose on production of these enzymes were monitored during cultivation. The highest production of polygalacturonase was achieved by Aspergillus niger during cultivation on sugar beet pulp moistened by mineral solution. The highest production of cellulase was achieved by Aspergillus niger during cultivation on wheat straw moistened by medium with glucose.