Thesis (doctoral)--University of Helsinki, 1986. / Includes bibliographical references (p. 31-38).

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Diploma thesis was aimed at studying of lipolytic activity of seven microorganisms, at which this activity was previously assumed or already confirmed. Theoretical part deals with the general characteristics of lipoyltic enzymes, specifics of microbial lipases and their industrial application, with an emphasis on food industry. Experimental part deals with the study of seven microorganisms and their lipolytic activity at the presence of a carbohydrate (glucose) or a lipid (canola oil) or both, as a source of carbon. For further testing were singled out Rhodotorula minuta nad Yarrowia lipolytica. This yeasts were subsequently tested for lipolytic aktivity at the presence of different vegetable oils (olive, sunflower, canola oil and waste cooking oil), nitrogen sources of organic and inorganic origin (urea, yeast extract, amonnium chloride, amonnium sulfate) and also a addition of detergent, which is commonly used in food industry or other food facilities.

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.

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.

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.

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.

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.

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.