The overall objective of this study was the optimization of a model enzymatic system in organic solvent media for the biosynthesis of selected phenolic lipid compounds. The model enzymatic system consisted of cinnamic acid and oleyl alcohol as substrates using commercial immobilized lipase (Novozym 435) from Candida antarctica. The experimental findings showed that an increase in the hydrophobicity of the solvent mixture and a decrease in the aw values of the reaction medium increased the initial enzymatic activity and bioconversion yield; the use of an iso-octane and butanone solvent mixture (85:15, v/v) and an initial aw of 0.05 resulted in an initial enzymatic activity of 192.7 nmol product/g enzyme/min and a corresponding bioconversion yield of 95.3% after a 16-day reaction period.

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mso-tstyle-colband-size:0 / mso-style-noshow:yes / mso-style-priority:99 / mso-style-parent:"" / mso-padding-alt:0cm 5.4pt 0cm 5.4pt / mso-para-margin-top:0cm / mso-para-margin-right:0cm / mso-para-margin-bottom:10.0pt / mso-para-margin-left:0cm / line-height:115% / mso-pagination:widow-orphan / font-size:11.0pt / font-family:"Calibri","sans-serif" / mso-ascii-font-family:Calibri / mso-ascii-theme-font:minor-latin / mso-hansi-font-family:Calibri / mso-hansi-theme-font:minor-latin / mso-bidi-font-family:"Times New Roman" / mso-bidi-theme-font:minor-bidi / mso-fareast-language:EN-US / } </style> <![endif]--> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (&lt / C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (&gt / C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to the</span><span style="font-size: 10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / mso-fareast-font-family:ArialOOEnc">&alpha / /&beta / hydrolase fold-3 domain protein of </span><i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Burkholderia </span></i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">sp. The RHgene34 protein </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into <i>Escherichia coli </i>Rosetta. <i>p</i>-Nitrophenol (<i>p</i>-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Kinetic analysis revealed that RHgene34 had a broad range activity on the <i>p</i>-Npesters, from C2 - C14. RHgene34 operates optimally at 45 &ordm / C, pH 9.0 and has a half life of 30 mins at 45 &ordm / C. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome.</span></p> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br /> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br />

Induction of extracellular lipases from Pseudomonas fragi CRDA 037 and Geotrichum candidum was used to increase lipase production in a shorter period of time. Induction was performed using edibles oils such as olive oil, canola oil, fish oil and butter fat. Fermentation trials of the microorganisms in the selected media were done in order to optimize the production of lipases. Optimal lipase activity was obtained in the presence of butter fat (1%, v/v) for P. fragi which was cultivated at 15°C during 48 h, and fish oil (0.75%, v/v) for G. candidum incubated at 27°C for 56 h. The induced lipase extracts of P. fragi and G. candidum obtained after these fermentation trials were purified by 3.7 and 5.9-fold respectively using ultrafiltration, while the non-induced fractions were purified by 2.3 and 5.1-fold, respectively. Activities were evaluated using p-nitrophenyl esters such as p-nitrophenyl laurate and p-nitrophenyl palmitate. (Abstract shortened by UMI.)

Lipase-catalyzed esterification and transesterification reactions of selected phenolic acids with lipids were investigated in organic solvent media. The esterification of linoleyl alcohol with dihydrocaffeic acid (DHA) in neat hexane medium resulted in highest esterification yield (EY) of 17% when a Candida antarctica lipase (Novozym 435) was used to catalyze the reaction. The use of co-solvents t-butanol and 2-butanone with hexane resulted in a dramatic increase in EY. The highest EY of 83% was obtained in hexane:2-butanone mixture of 85:15 (v/v) using Novozym 435; however lower EY (40%) was obtained when a lipase from Rhizomucor meihei (Lipozyme IM 20) was used. Increasing the amount of the co-solvent 2-butanone in the hexane:2-butanone mixture to 75:25 (v/v) resulted in a lower EY of 75% with Novozym 435; using the same enzyme, the esterification of a more unsaturated alcohol, linolenyl alcohol, with DHCA in the hexane:2-butanone mixture of 75:25 (v/v) resulted in EY of 76% which was similar to that obtained with linoleyl alcohol as lipid substrate. The esterification of DHCA and ferulic acid with linolenyl alcohol in the hexane:2-butanone mixture of 65:35 (v/v) resulted in an EY of 58 and 16%, respectively. Both linoleyl and linolenyl alcohols demonstrated mass action effects with EY of 99% in DHCA: fatty alcohol ratio of 1:8. Using a molar ratio of 1:2, the transesterification reactions of DHCA with trilinolein (TLA) and trilinolenin (TLNA) in hexane:2-butanone mixture of 75:25 (v/v) resulted in total transesterification yields (TYs) of phenolic lipids of 66 and 62%, respectively. The TYs of phenolic monoacylglycerols was higher than that of phenolic diacylglycerols for both TLA and TLNA transesterification reactions. A lower molar ratio of DHCA to TLA of 1:4 resulted in a lower TY of 53%. Using a molar ratio of 1:2, the TY of TLA and TLNA with ferulic acid in hexane:2-butanone mixture of 65:35 (v/v) was 16 and 14%, respectively. An equal molar transesterification reaction of DHCA with flaxseed oil, in a hexane:2-butanone mixture of 75:25 (v/v), resulted in the production of only phenolic monoacylglycerols (19%); however, decreasing the molar ratio resulted in the production of both phenolic mono and diacylglycerols. A molar ratio of DHCA to flaxseed oil (1:8) resulted in a TY of 76%, with 43 and 33% phenolic mono and diacylglycerols, respectively. Changing the solvent mixture of hexane:2-butanone from 65:35 to 85:15 (v/v) resulted in an increased in the TY of phenolic diacylglycerols from 24 to 55% with no significant effect on the TY of phenolic monoacylglycerols. The transesterification reaction resulted in a change in the composition of the C18:3 FA from 53% in the unmodified oil to 60 and 65% in the phenolic mono and diacylglycerols. Transesterification reaction of DHCA with fish liver oil in the solvent mixtures of hexane:2-butanone of 75:25 and 85:15 (v/v) resulted in TY of 56 and 65%, respectively. Transesterification in solvent: mixture of 75:25 resulted in a 40 and 16% TY of phenolic mono and diacylglycerols, respectively, whereas that in the solvent mixture of 85:15 (v/v) resulted in a 38 and 37% TY of phenolic mono and diacylglycerols, respectively. The structures of phenolic lipids of linoleyl and linolenyl alcohols with DHCA were confirmed by LC/MS analysis likewise for the phenolic mono and diacylglycerols from transesterification of DHCA with TLA and TLNA as well as flaxseed and fish liver oils. The phenolic esters of the fatty alcohols demonstrated radical scavenging properties similar to that of alpha-tocopherol but less than for DHCA; however, the phenolic lipids obtained with the use of TLA and TLNA as substrate as well as flaxseed and fish liver oil, demonstrated significant radical scavenging effects but less than that of alpha-tocopherol and DHCA.

Stuttgart, Univ., Diss., 2007.

Thesis (Ph. D.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 182-189).

University, Diss., 2002--Stuttgart. / Gedr. Ausg. im Inst. für Technische Biochemie der Univ. Stuttgart.

Orientador: Daniela Alonso Bocchini Martins / Banca: Ariela Veloso de Paula / Banca: Rubens Monti / Banca: Sandra Helena da Cruz / Banca: Roberta da Silva Bussamara Rodrigues / Resumo: Os fungos endofíticos são micro-organismos que vivem nos espaços intercelulares das plantas e são reconhecidos por seu potencial na produção de importantes produtos de interesse biotecnológico, tal como as enzimas. Estas vem sendo amplamente utilizadas nos setor industrial pois catalisam reações com maior especificidade que as formas convencionais de reações químicas, fazendo assim dos fungos endofíticos uma alternativa promissora na produção desses biocatalisadores. Desta forma, este trabalho teve como objetivo a prospecção de enzimas de interesse biotecnológico como lipase, esterase, epóxido-hidrolase, nitrilase, acilase, protease e amidase em fungos endofíticos e a avaliação da sua aplicação biotecnológica, com enfoque na utilização do extrato bruto contendo lipase em processos de biorremediação e transesterificação. Setenta e cinco fungos endofíticos foram submetidos à prospecção de atividade biocatalítica, onde 66 apresentaram atividade lipases/ esterases, 16 epóxido hidrolase, 24 acilases, 50 proteases, 10 nitrilase e 64 amidase. Foram encontrados fungos com satisfatória atividade biocatalítica frente a diferentes metodologias e substratos, mostrando a grande versatilidade dos endófitos na produção de enzimas. O fungo Mycosphaerella coacervata foi selecionado para avaliação da atividade e caracterização parcial da lipase extracelular em extrato bruto. O micro-organismo apresentou produção máxima da enzima quando cultivado por 48 horas a 28oC (486 U/L), e se manteve estável até 6 meses estocada a - 2oC. O extrato bruto contendo a enzima apresentou atividade máxima a 37oC em pH 8,0. A estabilidade térmica foi observada na faixa de 30 a 60oC (atividade residual acima de 80%) e sua estabilidade ao pH entre 6,0 e 9,0.(atividade residual superior a 80%). A enzima se manteve estável (atividade residual acima de 90%) na presença de acetona, metanol... / Abstract: The endophytic fungi are microorganisms that live in the intercellular spaces of plants and are known for their potential for the production of important products of biotechnological interest, such as enzymes. These have been widely used in industry because they catalyze reactions with greater specificity than conventional forms of chemical reactions, thereby making the endophytic fungus a promising alternative to produce these biocatalysts. Thus, this study aimed to prospect for enzymes of biotechnological interest as lipase, esterase, epoxide hydrolase, nitrilase, acylase, amidase and protease in endophytic fungi and evaluate their biotechnological applications, focusing on the use of the crude extract containing lipase in the transesterification process and bioremediation. Seventy five endophytic fungi were submitted to prospecting biocatalytic activity, where 66 showed lipase / esterase activity, 16 epoxide hydrolase, 24 acylases, 50 proteases, 10 nitrilase and 64 amidase. Fungi with satisfactory biocatalytic activity against different methodologies and substrates were found, showing the versatility of endophytes in the production of enzymes. The fungi Mycosphaerella coacervata was selected to evaluate the activity and partial characterization of extracellular lipase crude extract. The microorganism showed maximum enzyme production when grown for 48 hours at 28°C (486 U / L), and remained stable up to 6 months stored at -2°C. The crude extract containing the enzyme showed maximum activity at 37°C at pH 8.0. The thermal stability was observed in the range of 30 to 60°C (residual activity above 80%) and its stability at the pH between 6.0 and 9.0 (residual activity exceeding 80%). The enzyme was stable (residual activity above 90%) in the presence of acetone, methanol, ethanol, isopropanol and hexane at concentrations of 20 and 50%. Good stability was observed also in relation to different... / Doutor

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Alimentos, Florianópolis, 2011 / Made available in DSpace on 2012-10-25T21:32:35Z (GMT). No. of bitstreams: 1 290625.pdf: 2307335 bytes, checksum: 3e8ef1d7383b89281c74fd2e9dfb1b75 (MD5) / Policaprolactona (PCL) é um dos mais atrativos poliésteres alifáticos para aplicações biomédicas devido a sua biocompatibilidade, biodegradabilidade e compatibilidade com uma ampla faixa de polímeros. Diversas aplicações biomédicas podem ser encontradas na literatura sobre policaprolactona e seus copolímeros. Tradicionalmente, a PCL tem sido sintetizada por polimerização em massa, em solução ou em suspensão. Para qualquer destas técnicas é utilizado um iniciador organometálico na presença ou ausência de um co-iniciador contendo hidrogênio ativo. A poli(.-caprolactona) e seus copolímeros podem ser sintetizados quimicamente através do método da policondensação e do método da polimerização pela abertura do anel de ésteres cíclicos (lactonas, lactídeos e glicolídeos), o qual permite preparar poliésteres de alta massa molar. A utilização de enzimas como catalisadores na polimerização por abertura de anel vem se tornando uma alternativa bastante atrativa devido à polimerização ocorrer em condições mais amenas, principalmente no que concerne à temperatura. Desta forma, o objetivo do presente trabalho foi investigar a produção de poli(.-caprolactona) em dióxido de carbono supercrítico catalisada por enzima, especificamente a lipase comercial Novozym 435®. Os parâmetros empregados para avaliação da produção foram a massa molar e o índice de polidispersão obtidos na reação. Além disto, foi determinada a atividade da enzima após a reação com o intuito de verificar a possibilidade de seu reuso. Foram realizados dois planejamentos experimentais. No primeiro, as variáveis estudadas foram o binômio temperatura/pressão (35 a 65 °C/ 83 a 145 bar), a quantidade de enzima (1 a 10 %), a quantidade de água (0 a 2,5 %) e a agitação do meio (200 a 400 rpm). O diagrama de Pareto mostrou que apenas a variável quantidade de enzima foi significativa, no nível de 90 % de significância, para as faixas de condições estudadas. As massas molares médias numéricas variaram de 1050,4 a 7419,6 g/gmol. Não houve perda significativa de atividade enzimática após a reação com aumento de atividade em alguns casos, considerando-se o nível de significância de 90 % no diagrama de Pareto. O segundo planejamento avaliou os efeitos do deslocamento da faixa de estudo para níveis mais altos: pressão (150 a 200 bar), quantidade de enzima (5 a 15 %) e quantidade de água (0 a 4 %). Os resultados mostraram que o aumento dos níveis das variáveis operacionais não causou alteração estatisticamente significativa das reações para o nível de significância de 90 % no gráfico de Pareto. Nesse planejamento, as massas molares variaram de 1099,5 a 1463 g/gmol. Como a quantidade de água estava maior no sistema do segundo planejamento experimental (monômero, dióxido de carbono, enzima e, em alguns casos, água), uma das razões para a diminuição das massas molares em geral, poderia ser o efeito deletério da água no mecanismo da reação. A atividade enzimática para o segundo planejamento apresentou tendência a ser significativa no diagrama de Pareto para o nível de 90 % de confiança. Esse resultado também é considerável na diminuição das massas molares dos polímeros obtidos visto a importância da atividade da enzima no desencadeamento das reações. Os resultados obtidos nesse trabalho demonstram que o uso de catalisador enzimático e fluido supercrítico pode ser tão efetivo quanto às técnicas que empregam catalisadores metálicos e meios orgânicos, com a vantagem de não apresentarem toxicidade e não necessitar de tratamento para o descarte no ambiente. / Polycaprolactone (PCL) is one of the most attractive aliphatic polyesters for biomedical applications due to its biocompatibility, biodegradability and compatibility with a wide range of the polymers. Several biomedical applications can be found in the literature using poly(.-caprolactone) and their copolymers. Traditionally, the PCL has been synthesized by bulk polymerization, solution or suspension. In any of these techniques it is used organometallic initiators in the presence or absence of a co-initiator containing active hydrogen. The poly(.-caprolactone) and its copolymers can be chemically synthesized by the method of polycondensation and the method of polymerization by ring-opening of cyclic esters (lactones, lactide and glicolides), which allows to prepare high molecular weight polyesters. The use of enzymes as catalysts in the polymerization by ring-opening has become a very attractive alternative because the polymerization occurs in milder conditions, especially in relation to the temperature. The aim of this work was to synthesize poly(.-caprolactone) in supercritical carbon dioxide catalyzed by lipase, namely Novozym 435®. Enzymatic reactions were evaluated taking into account the molar mass and polydispersity index obtained. Besides, the enzyme activity was evaluated after polymerization reactions. Two experimental designs were carried out: in the first, the variables were binomial temperature/pressure (35 to 65 °C/ 83 to 145 bar), enzyme concentration (1 to 10 wt %), water content (0 to 2.5 wt %) and agitation (200 to 400 rpm). The Pareto diagram showed the enzyme concentration was significant (90 % confidence interval) in the experimental ranges investigated. The average molecular weight for the polymers ranged from 1050.4 to 7419.6 g/gmol. Enzymatic activity did not suffer significant changes for 90 % confidence interval. The second planning evaluated the effects of those variables at higher levels: pressure (150 to 200 bar), enzyme concentration (5 to 15 wt %), water content (0 to 4 wt %). Results showed that increasing levels of process variables did not cause a change for the statistical results of the reactions (90 % confidence interval). In these planning, the molecular weight ranged from 1099.5 to 1463 g/gmol. As the water concentration was higher in the second planning system, one of the reasons for the decrease of the masses in general, could be the deleterious effect of water on the reaction mechanism. The enzyme activity for the second design tended to be significant in the Pareto#s chart for the 90 % confidence interval. This result is also considerable for the decrease in the molecular weight of polymers whereas the importance of enzyme activity for the reactions. Results obtained in this study may be considered relevant as it is shown that the use of enzyme catalysts and supercritical fluids may be as effective compared to the traditional metal catalysts and organic medium, due to the advantages as no toxicity and does not require any treatment for disposal in the environment.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em Química. / Made available in DSpace on 2012-10-22T16:41:45Z (GMT). No. of bitstreams: 1 235685.pdf: 1733584 bytes, checksum: 300d8602199004129acc4b1fc6eca375 (MD5)