Pseudomonas fragi CRDA 037 was used as source of intracellular esterase, which remained attached to the cell membrane, and characterized. Several mechanical methods of disruption were used including glass beads (MSK), sonication, French press and combinations of these methods. The cellular debris were also treated with detergents such as CHAPS and Triton X-100 in the presence of chelating agent ethylenediaminetetra acetic acid (EDTA). The esterase activity remained in the cellular debris which was therefore use as a source of enzyme for kinetic studies. In the case of glass beads homogenization, the activity was found to decrease as a function of time of disruption. The results of chemical treatment showed that the esterase was characterized in terms of detergent and EDA action as well as substrate specificity. Triton X-100 and EDTA had no effect on the esterase activity and did not denature the enzyme. The substrate specificity with cells and cellular debris were carried out. The valeric acid was the best in term of esterase activity among fatty acids used in the study. (Abstract shortened by UMI.)

Thesis (doctoral)--University of Jyväskylä, 2006. / Includes bibliographical references. Also available on the World Wide Web.

Cells of Zymononas mobilis (ATCC 10988) were immobilised in calcium alginate beads and used to convert enzymatically hydrolysed cassava starch to ethanol. Optimum operating conditions were investigated in batch experiments. The ogtimum pH range and temperature were found to be 3-0 to 8.0 and 30 C, respectively. The maximum rates of glucose consumption and ethanol formation were obtained with an initial glucose concentration of 100 g/1. There was no fermentation inhibition below an initial ethanol concentration of 60 g/1. Ethanol productivity was the same using pure cassava hydrolysate or a medium composed of cassava hydrolysate, yeast extract and mineral nutrients. The immobilised Zymomonas mobilis cells were studied in a packed-bed reactor system operating under optimised parameters from the batch experiments. Volumetric ethanol productivities of 8.91 g/l.h and 22.5 g/l.h were obtained at 100% and 75% of glucose utilization, respectively; these productivities correspond to 1.5 times that of a free cell reactor when glucose utilisation was 100% and 3 times that of a free cell reactor when glucose utilisation was 75%. The maximum specific ethanol formation rate and the maximum specific glucose uptake rate were found to be 1.4 g/g.h and 2.8 g/g.h, respectively. The immobilised-cell reactor was operated continuously at a constant dilution rate of 0.2 h -1 for 20 days resulting in only a 20% loss of the original fermentative activity corresponding to an estimated half- life of 63 days. Based on experimental data, a mathematical analysis has been made and rate equations proposed.

Three Brazilian yeast strains, Saccharomyces cerevisiae 42 - F, Saccharomyces cereviaiae PLA 851 and Saccharomyces boulardli IZ 1904, all currently employed in the sugar fermentation industry, were evaluated with respect to their thermal tolerance and alcohol production kinetics. Best performance was found in Saccharomyces cerevisiae PLA 851 at temperatures up to 40 degrees (a common fermentation temperature in the Brazilian industry). This strain was further evaluated in chemostatic growth under sucrose limitation with biomass feedback on a 1 Litre scale in a specially constructed apparatus. At 30 degrees and 35 degrees under a dilution (growth) rate of 0.1 /h ethanol productivity increased by a factor of 2 with feedback and at 40 degrees by a factor of 3. The feedback factor (Beta) was 0.9. PLA 851 cells, heat - shocked at 45 degrees, resulted in a greater biomass productivity subsequently at 40 degrees coupled with a change in cell morphology. Highest ethanol productivity was found with 10% initial sucrose concentration at a dilution rate of 0.25 /h with feedback. Saccharomyces cerevisiae PLA 851 appears to be well adapted to the harsh physiological conditions in alcohol fermentations as currently practiced in Brazil.

Orientador: Ricardo Antunes de Azevedo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-03T21:30:01Z (GMT). No. of bitstreams: 1 Pereira_GuilhermeJoseGoncalves_D.pdf: 1076114 bytes, checksum: a6b5c1c85b9d597d9f61d04b0e962d8c (MD5) Previous issue date: 2004 / Doutorado

Submitted in fulfilment of the academic requirements for the degree of Master of Science in School of Molecular and Cell biology University of the Witwatersrand Johannesburg, South Africa / Esterases are hydrolytic enzymes that have many industrial applications. They are used in food, pharmaceutical, pulp and paper, cosmetics, biofuels and many other industries. This gives research of these enzymes major importance. Esterase genes received from CSIR Biosciences were cloned in E. coli DH5α cells. The plasmids carrying these genes were pET20b(+) for genes named Est1, Est2, Est3, Est4, Est5, Est6, Est7, Est8, Est9, Est10, Est12, Est13, Est14 and pET28a(+) for Est11. These plasmids were extracted from the cloning host and transformed into the expression host which was E. coli BL21. The cells were then induced for expression and the presence of the protein bands representing the products of expression were confirmed by running the crude enzyme extract on SDS-Page. The enzyme extracts were tested for activity using pNp-acetate. All 14 esterases were active and they were characterised in terms of pH optima, temperature optima and kinetics. The enzymes showed a pH range of 6.0 to 9.0 and temperature range of 30°C to 50°C. The enzymes were investigated for substrate specificity and they showed a greater preference for short acyl chain substrates over long acyl chain substrates. Further testing was done for activity of the enzymes using α-naphthylbutyrate and naphthol AS-D chloroacetate alongside lipases. A total of 87 enzymes were tested using these colorimetric assays and 36 of the enzymes were found to be active including all 14 esterases. These 36 enzymes were tested for use in enzymatic resolution of three different chemical compounds available as racemic mixtures. No success was observed for two of the compounds but one of them showed some enantioselectivity. This research will be furthered on at large scale to allow continued synthesis of potential HIV-1 protease inhibitors.

No description available.

No description available.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xvii, 122 p. : ill. Includes abstract. Includes bibliographical references (p. 46-53).

Orientador: Gabriela Alves Macedo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T14:07:21Z (GMT). No. of bitstreams: 1 Barros_Marciode_D.pdf: 1782603 bytes, checksum: fdeec5e629d7e54010f26836d322579b (MD5) Previous issue date: 2011 / Resumo: As esterases (carboxil-ester hidrolases; EC 3.1.1.3) e as lípases (triacilglicerol acilhidrolases; EC 3.1.1.3), são, coletivamente, conhecidas como enzimas lipolíticas, por apresentarem habilidade para hidrolisar ésteres de ácido carboxílico de cadeia curta e longa, respectivamente. As lipases catalisam a hidrólise de ligações éster na interface orgânica-aquosa, diferentemente das esterases que catalisam a hidrólise de ligações éster de substratos em meio aquoso. O objetivo deste trabalho foi avaliar a atividade de esterase nos extratos brutos das sementes de soja (Glycine Max L.), sorgo (Sorghum bicolor L.) e cacau (Theobroma cacao); selecionar as sementes que apresentam maior atividade esterásica, e avaliar as características bioquímicas das enzimas brutas e purificadas. Foi observada atividade lipolítica e esterásica nas amêndoas de cacau e esterásica nas sementes de soja e sorgo. Foram avaliadas diferentes soluções para a extração da esterase de soja e observou-se que a solução de CaCl2 0,01M mostrou-se mais eficiente na extração da enzima sendo obtido atividade igual a 1,83 U.mg-1. No estudo da concentração da esterase de sorgo por fracionamento com sulfato de amônio foi obtido maior atividade (188,30 U.mg-1) utilizando 60% de saturação do sal. Na precipitação da esterase de soja com sulfato de amônio a enzima apresentou baixa estabilidade. Em relação aos parâmetros bioquímicos, a esterase de sorgo apresentou atividade ótima na temperatura de 40ºC, porém apresentou baixa estabilidade nesta temperatura, o pH ótimo de atividade da enzima foi pH 8 e a enzima demonstrou ser estável em toda faixa de pH estudada (3 a 10), quanto ao substrato a enzima demonstrou maior afinidade por pNPB cuja atividade foi 70,82 U.mL-1. Quanto a esterase de soja, as preparações bruta e purificada apresentaram o mesmo comportamento apresentando atividade ótima em pH 8,0 e a 40°C, estabilidade na faixa de pH 6,5 a 10 e estabilidade até 50°C durante 60 minutos. O composto polietilenoglicol de peso molecular 0,4; 6 e 10kDa ativou respectivamente 140, 170 e 140% a esterase de soja bruta. A esterase de soja purificada apresentou um aumento de 11 vezes na atividade, com um Km de 0,85 µM e Vmax 31,5 U.mL-1. As características apresentadas pelas esterases de soja e sorgo as qualificam para aplicação industrial / Abstract: Esterases (carboxyl ester hydrolases; EC 3.1.1.3) and lipases (glycerol ester hydrolases EC 3.1.1.3) are enzymes capable of hydrolyzing short and longchain carboxylic acid esters, respectively. The lipases hydrolyze the ester bonds of their water-insoluble substrates at the organic-aqueous interface, differently from the esterases, that hydrolyze the ester bonds of their soluble substrates in the aqueous medium. The objective of this work was to evaluate the presence of esterases and/or lipases in crude extracts of soybean (Glycine Max L.), sorghum (Sorghum bicolor L.) and cacao (Theobroma cacao) seeds, select the seeds with the highest esterase activities, and study the biochemical properties of the crude and purified enzymes. Lipolytic and esterase activities were found in the cacao seeds, but only esterase activity in the soybean and sorghum seed crude extracts. Different solutions were evaluated to extract the soybean esterase, and it was found that 0.01M CaCl2 was the most efficient, obtaining activity of 1.83 U.mg-1. In the fractional precipitation process with ammonium sulfate aimed at concentrating the sorghum esterase, the highest activity (188.30 U.mg-1) was obtained with 60% saturation of the salt. However, when ammonium sulfate was used to concentrate the soybean esterase, low enzyme stability was observed. With respect to the biochemical parameters, the sorghum esterase showed optimum activity at pH 8 and a temperature of 40°C, but showed low stability at 40°C although it was stable in the entire pH range studied (3 to 10). With respect to the substrates, it showed greatest affinity for short-chain fatty acids (pNPB), with activity of 70.82 U.mL-1. The crude and purified preparations of the soybean esterase showed the same behavior, with optimum activity at pH 8 and 40°C, and were stable at pH values between 6.5 and 10 and at temperatures up to 50ºC for 60 minutes Polyethylene glycol with molecular weights of 0.4, 6 and 10kda activated 140, 170 and 140% of the enzyme activity, respectively. The soybean esterase was purified using DEAESepharose ion exchange column chromatography followed by passage through a Sephadex G-100 gel column and ultra-filtration, leading to an overall purification of 17.6 fold, with a Km of 0.85 µM and Vmax of 31.5 U.mL-1. The biochemical characteristics presented by the soybean and sorghum seed esterases qualified them for industrial applications / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos