Lipases são enzimas que hidrolisam especificamente óleos e gorduras, podendo ser de grande interesse para vários segmentos industriais como têxtil, cosméticos, alimentícios e tratamento de efluentes com elevada carga de gordura. O objetivo do presente trabalho foi avaliar a produção de complexos enzimáticos lipolíticos produzidos por microrganismos isolados de um sistema de tratamento biológico de efluentes de um hotel, visando obter lipases com potencial para emprego em processos biotecnológicos. Para a seleção do microrganismo lipolítico foram utilizados 65 isolados bacterianos oriundos do efluente bruto e tratado da wetland e da caixa de gordura do restaurante do hotel. A produção de lipase foi testada em placa em meio de cultivo acrescido de azeite de oliva e rodamina B, incubados a 25°C e 30ºC por 24h-48h e as placas observadas sob luz UV 350 nm. Deste total de isolados, sete oriundos da wetland e 22 isolados da caixa de gordura foram positivos para lipase, pois apresentaram halos fluorescentes alaranjado. Os isolados foram testados novamente em placa, porém numa condição mais estressante em termos de nutrientes para induzir a utilização do azeite e assim selecionar as melhores produtoras de enzimas Desses, 12 isolados apresentaram atividade lipolítica e aquele que apresentou halo mais evidente e maior foi selecionado para ensaios com crescimento em cultura submersa em agitador orbital e biorreator em três substratos diferentes (azeite de oliva, óleo de semente de uva e óleo de canola) para a determinação da atividade enzimática. As bactérias lipolíticas identificadas bioquimicamente foram do gênero Enterobacter, Acinetobacter, Pseudomonas, Klebsiella e Burkholderia. Os ensaios em cultura submersa em biorreator nos três substratos avaliados apresentaram a máxima produção de lipase após 12 horas de cultivo; azeite de oliva 0,358 U/mL.min-1, óleo de semente de uva 0,352 U/mL.min-1 e óleo de canola 0,348 U/mL.min-1. Em cultivo submerso em agitador orbital, o meio de cultivo na presença de Tween 80, apresentou melhor atividade enzimática que o meio de cultivo sem a presença do emulsionante. A identificação molecular do isolado apresentou identidade com o gênero Acinetobacter baylyi. A análise por SDS-PAGE sugere uma lipase com massa molecular de 35 kDa. / Lipases are enzymes that specifically hydrolyze oils and fats and may be of great interest to various industrial segments such as textiles, cosmetics, food and effluent treatment with the high fat load. The aim of this work was to evaluate the production of lipolytic enzymatic complexes produced by microorganisms isolated from a biological treatment system of effluents of a hotel, aiming to obtain lipases with potential for use in biotechnological processes. To select the best lipolytic microorganism, we used 65 bacterial isolates, recovered from the raw and treated effluent of the wetland system and from the fat tank of the hotel restaurant. Lipase production was evaluated in culture medium supplemented with olive oil and rhodamine B, incubated at 25°C and 30°C for 24h-48h. The grown colonies were observed under UV light at 350 nm. Out of that, seven isolates from wetland and 22 isolated from the fat tank were positive for lipase as they exhibited orange fluorescent halos. The isolates were again seeded on medium with a stressful nutrient condition to induce the use of olive oil and thus select the best enzyme producers Of these, 12 isolates presented lipolytic activity, and the one that showed the most evident halo was selected for assays in submerged culture in an orbital shaker and bioreactor using three different substrates (olive oil, grape seed oil, and canola oil). The lipolytic bacteria identified were of the genus Enterobacter, Acinetobacter, Pseudomonas, Klebsiella, and Burkholderia. The assays run in the bioreactor showed the maximum lipase production after 12 hours of culture with the three substrates evaluated; 0,358 U/mL.min-1 in olive oil, 0,352 U/mL.min-1 grape seed oil, and 0.348 U/mL.min-1 canola oil. The results obtained in an orbital shaker showed better enzymatic activities in the presence of Tween 80 on the culture medium. The molecular identification showed identity with the genus Acinetobacter baylyi. Analysis by SDS-page suggests a lipase with a molecular mass of 35 kDa.

Made available in DSpace on 2017-07-10T17:48:02Z (GMT). No. of bitstreams: 1 Gilberto_Henrique_Simoes.pdf: 609344 bytes, checksum: c392f5329a574e72a4a5033766bd505a (MD5) Previous issue date: 2014-04-15 / The complexity and diversity of the dairy production chain in the country are due to the different types of dairy production systems and their specific characteristics, among them, the spatial and cultural differences found across the country, making it difficult to standardize the production, planning, technicization and especially the microbiological quality of the raw material in the dairy chain. In order to illustrate the dairy production systems, the managements, critical points of contamination, isolation and identification of the main factors involved in the contamination in Marechal Cândido Rondon - PR, some experiments were conducted. Initially it was performed a selection among 735 dairy production systems for semi - structured guides (production characteristics and general management) and cluster analyzes, which formed five distinct and homogeneous groups of dairy production systems. Only 10% of properties were selected to represent each group, totalizing 73 properties. Subsequently it was analyzed the milk cooling tanks for the somatic cell count (SCC) and total the bacterial count (TBC). After the counts, only 35 properties were selected for the research because they showed contaminations rates above current limits for SCC and TBC according to the MAPA regulation (IN 62). After the formation of the systems, collections related to the research were made, which consisted of the semi-structured questionnaire guide (hygienic-sanitary management), milk collection of the tank and swabs of critical points of contamination in milk production (hand of the milker, milking machine and cooling tank). The experiment was proceeded with microbiological analysis and quantification of Staphylococcus sp, hand of the milker, milking machine, tank and milk 9.6 x103, 2.2 x104, 1.4 x104 and 3.8 x103 CFU/mL respectively, and quantification, isolation and identification of proteolytic and lipolytic bacterias, in which the agents of highest incidence were Escherichia coli, Escherichia fergusoni, Yersinia enterocolitica and Klebsiella oxytoca both with 8.6% frequency in the different dairy production systems studied / A complexidade e diversidade da cadeia produtiva leiteira existente no país devem-se aos diferentes tipos de sistemas produtivos leiteiros e as características próprias que os envolvem, entre elas as diferenças espaciais e culturais encontradas em todo o território nacional, dificultando assim uma padronização na produção, planejamento, tecnificação e principalmente da qualidade de produção microbiológica da matéria-prima na cadeia leiteira. Afim de caracterizar os sistemas de produção leiteiros, os manejos empregados, pontos críticos de contaminação e de isolamento e identificação dos principais agentes envolvidos nas contaminações da microrregião de Marechal Cândido Rondon PR, estudos foram realizados. Inicialmente efetuou-se seleção dentre 735 sistemas de produção leiteiros, nestes foram aplicados guias semiestruturados para levantamento de características de produção e manejo produtivo. A partir destes resultados foram empregadas técnicas de formação de clusters. Foram definidos cinco grupos homogêneos e distintos. Em cada grupo, 10% dos casos foram selecionados, totalizando 73 sistemas produtivos leiteiros. Posteriormente analisou-se o leite dos tanques de resfriamento para contagem de células somáticas (CCS) e contagem bacteriana total (CBT). Após as contagens, 35 propriedades foram selecionadas paras as pesquisas, pois obtiveram contaminações acima dos limites vigentes de CCS e CBT de acordo com IN 62 do MAPA. Após a formação dos sistemas sucedeu-se coletas referentes às pesquisas, que consistiram de questionário semi-estruturado, contemplando variáveis relativas ao manejo higiênico-sanitário, bem como, coleta de leite do tanque e swabs de pontos críticos de contaminação na produção leiteira, tais como: mão do ordenhador, ordenhadeira e tanque de resfriamento. Procedeu-se com análises microbiológicas e a quantificação de Staphylococcus sp da mão-do-ordenhador, ordenhadeira, tanque e leite 9,6x103, 2,2x104, 1,4x104 e 3,8x103 UFC/mL respectivamente; e quantificação, isolamento e identificação de bactérias proteolíticas e lipolíticas, nos quais os agentes de maior incidência foram a Escherichia coli, Escherichia fergusoni, Klebsiella oxytoca e Yersinia enterocolitica ambos com 8,6% de frequência nos diferentes sistemas de produção leiteiros estudados

As enzimas lipolíticas vêm atraindo atenção no mercado global devido ao enorme potencial biotecnológico, como: na formulação de detergentes; na indústria de couro; produção de cosméticos, fármacos, aromas, biodiesel, etc. O objetivo deste trabalho foi prospectar genes codificadores de enzimas lipolíticas em biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel. A seleção foi feita pela atividade lipolítica através do cultivo dos clones em placa de petri e a avaliação foi pela observação de halo ao redor da colônia, sendo positiva para 30 clones dentre os quais dois se destacaram. Estes dois clones foram selecionados e subclonados. Os DNAs das sub-bibliotecas foram sequenciados, gerando um contig completo para cada clone. Através do ORF Finder foi identificado cinco ORFs de esterase/lipase, dentre as quais uma alcançou 58% de identidade com uma bactéria não cultivável. As árvores filogenéticas indicam que duas ORFs são similares à família IV das enzimas lipolíticas, enquanto que as outras três ORFs à família V. / Lipolytic enzymes have been attracting global market attention because they show enormous biotechnological potential. The present work was done as an attempt to find genes which codify lipolytic enzymes in a metagenomic library composed of diesel oil degradation microbe consortia. Clones were selected according to lipolytic activity and were then evaluated after cultivation in Petri dishes by observation of halo formation around the colonies. 30 clones produced halo formations and were identified as positives, two of which showed prominent results. These two were then selected and sub cloned. DNA from the sub libraries was sequenced, generating a complete contig for each clone. Using the ORF Finder five esterase/lipase ORFs were identified, with one of these attaining 58% of identity to a non cultivatable bacteria species. Assessment of the cladograms showed that two ORFs were similar to lipolytic enzyme family IV, while the other three ORFs were similar to family V.

This diploma thesis is focused on controlled production of lipolytic enzymes, bioactive substances and lipids by carotenogenic yeasts. Theoretical part deals with characterization of lipolytic enzymes, carotenoids, lipids and their properties, possibility of production and application. In experimental part the enzymes, carotenoids and lipids were produced by red yeasts Rhodotorula mucilaginosa, Cystofilobasidium macerans and Sporidiobolus salmonicolor by submerged cultivation in mineral medium with different additions: glucose, glycerol, fat, fat with glucose, fat with polysorbate 80, fat with glycerol, fat with polyethylene glycol, fat with higher and lower addition of palmitic acid, enzymatic fat hydrolysate, acidic hydrolysate a basic hydrolysate. The activity of extracellular lipase was monitored in medium after 96-hour cultivation. Concentration of -carotene, total carotenoids, ergosterol and ubiquinone was determined by HPLC, concentration of fatty acids and amount of fat by GC. Production had differed depending on used yeasts and substrate. As the best producer of carotenoids Cystofilobasidium macerans was found, ergosterol was highly produced by Sporidiobolus salmonicolor. The production of ubiquinone was almost equivalent in all yeasts and lipolytic activity was the highest in Sporidiobolus salmonicolor. The patricular medium sample with high lipolytic activity was further separated and analysed by ultrafiltration and PAGE-SDS electrophoresis. This diploma thesis was done within the international project ,,LipoFungi“.

Lipases are enzymes catalyzing primarily the hydrolytic cleavage of triacylglycerol bonds. The production of lipolytic enzymes is known in many microorganisms, especially those who are able to utilize a fatty carbon substrate. Some genera of carotenogenic yeasts are characterized by this ability. Carotenogenic yeasts are characterized primarily by the formation of intracellular carotenoids, lipids and lipid-soluble substances. In addition to these metabolites, they may also produce some biosurfactants. This work deals with the production of extracellular lipolytic enzymes and biosurfactants by carotenogenic yeasts Rhodotorula glutinis, Cystofilobasidium macerans, Rhodotorula mucilaginosa and Sporidiobolus pararoseus cultivated mainly on animal waste fat at various C/N ratios (13, 25, 50, 100). Lipase activity was detected in all strains studied. Enzyme activities were measured by spectrophotometric method. Lipase induction has also been observed during cell growth, where several peaks of lipase activity have been reported, suggesting cell-associated lipase and lipase secreted into the environment. Lipase activities have also been found in cultures on glucose and glycerol carbon substrates. Further, the molecular characterization of lipolytic enzymes was performed using polyacrylamide gel electrophoresis. The formation of biosurfactants is to some extent formed by all strains. In particular, the biosurfactants of C. macerans and S. pararoseus yeast have emulsifying and solubilizing properties. Simultaneously with the production of lipase and biosurfactants, the production of characteristic high value added intracellular metabolites in S. pararoseus and R. mucilaginosa was evaluated too.

Diploma thesis was aimed at testing of five commercial products for separators of lipids. Performed study should to shown that in produts are presented lipase-produducing microoganisms, event. that in products are also lipolytics enzymes as addition. The products was cultivated by solid-state and submerged fermentations and subsequently was tested their properties as is lipolytic activity, the amount of free fatty acids etc. Solid-state fermentation used Tributyrin Agar Base where Tributyrin was used as a substrate. Submerged fermentation used special prepared medium where was olive oil. Methods using in this study served for detection and qualification of lipase-produducing microoganisms in individual products. For detection of lipase-produducing bacterial spieces was used Spirit Blue Agar. In this case was used Tributyrin and Tween 80 as a substrate. Lipolytic activity was measured by spectrophotometry and degradation of lipids was measured titrimetry. Performed study demonstrated that all tested products have ability to degradation of lipids.

The thesis deals with the study of the different yeast strains from the point of view of extracellular lipolytic enzyme production. First part of this work consisting of appropriate yeasts was developed within study interships in Slovak Academy of Sciences, department of Glycomics in Bratislava. From ten given strains three yeasts such as Pseudozyma fusiformata, Meyerozyma guilliermondii, Yarrowia lipolytica were chosen, these strains showed the highest lipolytic activity and cell growth on basal medium with Tween 80. These yeasts were used for optimization of cultivation conditions and characterization of lipolytic enzymes. The yeasts were cultivated on media with different carbon sources, which appeared to be a most suitable medium the basal medium with Tween. Tween acted as and inducer of lipase production. The substrate specificity was determined using three p-nitrophenylester substrates with varying sizes of the fatty acid site chains. The results showed that tested lipases are probably triacylglycerol-acyl-hydrolases which has the highest activity towards in the water insoluble substrates with medium long chains. The pH optimum and temperature optimum were measured. The results showed that the tested lipases had the highest activity in neutral and mild acid region around 30°C. By measuring of thermal stability has been demonstrated that extracellular lipases are relatively thermostable enzymes. Afterwards the storage stability was measured for 5 weeks when supernatant was kept in fridge at 4°C and in freezing box at -20°C. The results showed that in both cases tested lipases exhibited high storage stability which allows to store the samples without loss of activity for a longer time. Finally, the results of lipolytic and proteolytic activity, cell growth and pH of the medium of yeast Y. lipolytica were compared between the batch cultivation in L-tubes with the continual cultivation in the bioreactor. The highest lipases production was achieved in bioreactor due to the setting conditions of the continual proces to regulate the production and enzymatic stability.

This work deals with the problem of microbial degradation of the waste materials from food industry. This work is focused on the production of technological significant enzymes producing by microorganisms, which were able to use the waste as a sole carbon source. In the first part of this work, the attention was focused on the production of pectolytic enzymes. This part was made within study interships in Slovak Academy of Sciences, department of Glycomics in Bratislava. The grape pomace as the waste form winegrowing was used as a sole carbon source for microbial growth and enzymes production. The production of pectolytic enzymes was tested on this waste. After screening the most suitable microorganisms was chosen with the highest production of polygalacturonase activity. Produced enzymes were isolated by extraction techniques, purified and then identified proteomically. The aim of the second part of this work was the waste water treatment containing lipids and lipolytic enzymes. The reason was the cooperation with the company constructing grease traps. The characterization of supplied commercial preparations was the subject of this work and the other reason was the characterization of conditions for lipases secreting by microorganisms, identification of microorganisms present in the commercial preparation and testing of new microbial cultures for the development of new preparation for the grease traps.

In this thesis, optimization of production of lipolytic enzymes by submerzed cultivation of Bacillus subtilis (BS) was studied. Production of lipolytic enzymes was tested in three nutrient media, which differed mainly in main sources of carbon, respectively of nitrogen. The first medium contained mainly extract from calf brain and beef heart (BHIB), the second medium contained peptone and yeast extract (NB) and the third one contained peptone and yeast extract with the addition of 2% (w/v) glucose (NBG). The highest lipolytic activity (0.0784 Uml-1) was measured in NBG medium. Maximum of lipolytic activity was observed before the end of the exponential phase of BS growth in all the media. Temperature optimum in NBG medium was determined from 30 to 50 °C, pH optimum in the range of 5 to 11 and subsequently the temperature stability of lipolytic enzymes produced by the BS was estimated. The activity value was determined spectrometrically using p-nitrophenyllaurate as a substrate. Produced lipolytic enzymes showed maximum activity at 37 °C in the alkaline pH of 8.0. Measurement of temperature stability showed that lipolytic enzymes are relatively thermostable enzymes retaining 100 % of the activity even after 1 hour of cultivation at 30 - 50 °C. The presence of 1% (w/v) olive oil in medium NBG caused a decrease in lipolytic activity by 65 % as well as in pH from 6.5 to 5.4 after 14 days of cultivation. After substitution of glucose by fructose in medium NBG, lipolytic activity showed comparable values during the first week of cultivation. On the other hand, the decrease of lipolytic activity by 29 % in the medium with fructose was observed after 14 days of cultivation. A procedure for the identification of lipolytic enzymes of BS by peptide massfingerprinting was developed to understand the potential of synthetic polyester - poly(e-caprolactone) as a lipase inductor. Degradation study of commercial polyester poly(e-caprolactone) was carried out by submerged cultivation of Bacillus subtilis in NBG medium at initial pH 7.0 and 30 °C for 14 days. PCL (Mn = 10,000, Mw = 14 000) was studied in the form of films (1.0 x 1.0 cm), which were prepared by melt-pressing, rapid cooling of the melt to 4 °C and evaporation of the solvent from 2 % dichlormethane solution. The evaluation of the films shown occurrence of weight loss (7.8 - 17.0 wt.%) together with the formation of numerous holes and cracks in the sample surface in relation to the method of the films preparation. Lipolytic activity values increased by 9 - 17 % in the degradation media compared to control samples. Densitometric monitoring showed also higher increase in cell mass in the degradation medium compared with control samples. Based on the results obtained, the degradation process induced by BS could be suggested.

As caprilinas são acilgliceróis de cadeia média com diversas aplicações nos campos alimentício, farmacêutico e cosmético. A esterificação direta é um processo importante para a síntese de caprilinas, dado que o glicerol é subproduto da produção de biodiesel e que a tricaprilina, material de partida para a glicerólise e hidrólise, não é um composto abundante naturalmente. As reações existentes para a síntese de acilgliceróis podem ser catalisadas por via química ou enzimática, sendo esta última superior em diversos aspectos. Dois dos principais contrapontos à utilização de enzimas são seu alto custo e dificuldade de recuperação. A imobilização de enzimas é uma das maneiras existentes para contornar estas questões. A maioria dos estudos científicos relativos à obtenção destes compostos refere-se aos acilgliceróis de cadeia longa. Portanto, é necessário aprimorar a esterificação direta entre o glicerol e ácido caprílico catalisada por lipase, através da compreensão da cinética reacional e da influência dos parâmetros reacionais. A existência de uma metodologia de separação, identificação e quantificação simples e eficaz também é importante, tornando as pesquisas relacionadas às caprilinas mais rápidas e menos complicadas. Desta maneira, este trabalho consistiu de três etapas principais: (1) a imobilização de lipases em resinas de troca aniônica, visando reduzir os custos em relação à lipase imobilizada comercial; (2) o desenvolvimento de um método de cromatografia em camada delgada capaz de quantificar as caprilinas formadas; (3) o estudo da reação de esterificação para a obtenção de caprilinas em regime descontínuo e descontínuo-alimentado. A resina DOWEX® 1X2-400 foi a que apresentou a melhor eficiência de imobilização (EI) da Palatase®. Sua atividade lipolítica foi apenas 12,7% inferior à versão imobilizada comercial da lipase, a Lipozyme®RM IM, indicando a viabilidade de substituição desta última. O desenvolvimento de método de cromatografia em camada delgada resultou em relações lineares para a detecção e quantificação de ácido caprílico, monocaprilina, dicaprilina e tricaprilina com R2 e R2pred de 99,3%/98,73%; 98,9%/97,6%; 99,8%/99,8% e 99,7%/99,5%, respectivamente, e com valor-p de 0,000. As reações em regime descontínuo foram realizadas seguindo planejamento fatorial completo, no qual foram variadas a temperatura (30 ou 70°C), a proporção molar entre os reagentes (1:1 ou 3:1 - ácido caprílico : glicerol) e o tempo reacional (6 ou 10h). A análise de regressão do rendimento reacional expresso em porcentagem de consumo de ácido caprílico indicou que, nestas condições, os fatores relevantes para o resultado final são a temperatura e a proporção molar, bem como a interação entre eles. As melhores condições para a obtenção de mono e dicaprilinas foram, portanto, de 30ºC e razão molar de 1:1. O estudo da cinética da reação indica que 40,4% do ácido caprílico são consumidos na primeira hora e que o consumo máximo ocorre na terceira hora de reação. Houve uma queda drástica no rendimento reacional, quando a reação foi conduzida em regime descontínuo-alimentado, provavelmente devido à menor eficiência catalítica da lipase devido ao baixo teor de água na primeira hora de reação. / Caprylins are medium chain acylglycerols that find several applications in food, pharmaceutical and cosmetic fields. Direct esterification is an important route for the synthesis of caprylins, since glycerol is a byproduct of biodiesel production and tricaprylin, the starting material of glycerolysis and hydrolysis, is not a naturally abundant compound. The glycerides synthesis can be catalyzed by either chemical or enzymatic routes, the latter being superior in several aspects. The biggest issues regarding the use of enzymes are their high cost and difficult recovery. Using immobilized enzymes can minimize these concerns. Most of scientific studies concerning the obtainment of these compounds address to long chain acyglycerols. Thus, the improvement of lipase catalyzed esterification of glycerol and caprylic acid is necessary, by understanding the reaction kinetics and the role of reaction parameters. A simple and effective method of separation, identification and quantification of caprylins is also a relevant contributing factor for easier and faster researches. Therefore, this work consisted of three main steps: (1) immobilization of lipases on anion exchange resins in order to produce a cheaper alternative to imported commercial immobilized lipases; (2) development of a thin layer chromatographic method capable of quantifying caprylins; (3) studying the obtainment of caprylins by direct esterification in batch and fed batch systems. DOWEX® 1X2-400 resin presented the best immobilization efficiency (IE) results. Additionally, its lipolytic activity was only 12.7% lower than the commercial Lipozyme® RM IM, suggesting that Lipozyme\'s substitution is feasible. Regarding the developing of a thin layer chromatography method, linear correlations were obtained for detection and quantification of caprylic acid, monocaprylin, dicaprylin and tricaprylin, with a R2 and R2pred of 99,3%/98,73%; 98,9%/97,6%; 99,8%/99,8% e 99,7%/99,5%, respectively, with a p-value of 0,000. The batch reactions were carried out according to a complete factorial design, in which the temperature (30 or 70ºC), the molar ratio between the reagents (1:1 or 3:1) and reaction time (6h or 10h) were varied. The regression analysis of reactional yield expressed in terms of percentage of caprylic acid consumption indicated that only the temperature, molar-ratio and their interaction were important for reaction yields, within the studied conditions. The best conditions related to the obtainment of mono and dicaprylins were 30ºC and molar ratio of 1:1. The reaction kinetics indicates that 40.4% of the caprylic acid is consumed within one hour of reaction and that the maximal consumption was achieved in the third hour. A drastic drop in reaction yield was observed when the fed batch mode was adopted, probably due a lower catalytic efficiency presented by lipase as a result of the lower water content in the first hour of reaction.