L’hyperchylomicronémie familiale est un trait monogénique caractérisé par un taux de triglycérides plasmatiques à jeun supérieur à 10 mmol/L (la normale étant de 1,7 mmol/L). L’hyperchylomicronémie familiale est le plus souvent causée par une déficience dans le gène LPL (pour lipoprotéine lipase). La déficience en lipoprotéine lipase (LPLD) est aussi associée à un risque élevé de pancréatite. La pancréatite en soi est reconnue comme un trait complexe génétique dont plusieurs gènes sont associés à sa susceptibilité. Étant donné l’expression variable de la pancréatite chez les patients LPLD, les résultats de ce mémoire présentent certains facteurs génétiques pouvant être responsables du risque de l’expression de la pancréatite aigüe récurrente chez les sujets LPLD. L’analyse par séquençage des régions codantes et promotrices des gènes CTRC (pour « Chymotrypsin C ») et SPINK1 (pour « Serine protease inhibitor Kazal type 1 ») a été effectuée chez 38 patients LPLD et 100 témoins. Ces deux gènes codent pour des protéines impliquées dans le métabolisme des protéases au niveau du pancréas et ont déjà été associés avec la pancréatite dans la littérature. Notre étude a permis d’identifier une combinaison de deux polymorphismes (CTRC-rs545634 et SPINK1-rs11319) associée significativement avec la récidive d’hospitalisations pour douleur abdominale sévère ou pour pancréatite aigüe récurrente chez les patients LPLD (p<0,001). Ces résultats suggèrent que le risque de récidive de pancréatite chez les patients LPLD peut être influencé par des variants dans des gènes de susceptibilité à la pancréatite. L’identification de biomarqueurs génétiques améliore la compréhension des mécanismes physiopathologiques de la pancréatite chez les patients LPLD ce qui, par conséquent, permet de mieux évaluer et caractériser les risques de pancréatite afin d'adapter un plan d'intervention préventif pour ces patients. / Familial hyperchylomicronemia is a monogenic trait characterized by an increased fasting plasma triglyceride levels ≥ 10 mmol/L (normal is 1.7 mmol/L). Familial hyperchylomicronemia is most often caused by a deficiency in the LPL gene. Lipoprotein lipase deficiency (LPLD) is also associated with an increased risk of pancreatitis. Pancreatitis is recognized as a complex genetic trait and several genes are associated with its susceptibility. Considering the variable expression of pancreatitis in LPLD patients, results of this manuscript demonstrate that genetic factors may be responsible of the increased risk of recurrent acute pancreatitis episodes in LPLD subjects. The sequencing analysis of the coding and promoters regions of CTRC gene (for Chymotrypsin C) and SPINK1 gene (for Serine protease inhibitor Kazal type 1) was performed. These two genes encode proteins involved in the metabolism of the pancreas proteases and have been associated with pancreatitis in literature. A combination of two polymorphisms (CTRC-rs545634 and SPINK1-rs11319) have been identified and associated with recurrent hospitalizations for severe abdominal pain or recurrent acute pancreatitis in LPLD patients (p <0.001). These results suggest that the risk of recurrent episodes of pancreatitis in LPLD patients may be influenced by variants in susceptibility genes. The identification of genetic biomarkers improves the understanding of the pathophysiological mechanisms of pancreatitis in LPLD patients which therefore helps to assess and characterize the risk of pancreatitis to adapt preventive intervention plan for these patients.

Neste trabalho realizou-se uma triagem com os fungos de origem marinha Trichoderma sp Gc1, Penicillium miczynskii Gc5, Penicillium raistrickii Ce16 e Aspergilus sydowii Gc12 para catalisar a abertura do (RS)-2-(benziloximetil)oxirano (2). O melhor resultado foi obtido com o fungo Trichoderma sp Gc1, pois forneceu o (R)-(-)-2-(benziloximetil)oxirano (2) com excesso enantiomérico de 60 % e rendimento isolado de 39 %; o diol (S)-(+)-1,2-propanodiol-3-fenilmetóxi (2a) com excesso enantiomérico de 32 % e rendimento de 19 %. Posteriormente otimizou-se as condições experimentais com o epóxido 2 e o fungo Trichoderma sp Gc1, variando-se a massa de biocatalisador, o meio de cultura e o tempo de reação. Os melhores resultados sob essas condições foram aplicadas para os epóxidos 3-5 fornecendo o (S)-(+)-2-[4-metoxifenoxi)metil]oxirano (3a), (S)-(+)-2-(propeniloxi)oriano (4), (R)-(+)-1-alilóxi-2,3-propanodiol (4a) e o (-)-9-deceno-1,2-diol (5a). Nesses estudos embora ocorreu a abertura seletiva dos epóxidos com as células totais do fungo Trichoderma sp Gc1, não obteve-se altas purezas enantioméricas dos produtos. Ainda nesse trabalho realizou-se a síntese das cloroidrinas racêmicas, a (RS)- 1-cloro-2-propanol- 3-fenilmetóxi (2b), (RS)- 1-cloro-2-propanol- 3-(4-metoxifenóxi) (3b) e (RS)- 1-alilóxi-3-cloro-2-propanol (4b) em bons rendimentos e uma metodologia sintética ambientalmente apropriada, pois os compostos foram preparados em meio aquoso na presença de íons cloreto. Em seguida realizou-se uma resolução enzimática da (RS)-1-alilóxi-3-cloro-2-propanol (4b) com a lipase de Candida antarctica onde obteve-se a clorodrina 4a (e.e. 72 %) e o seu correspondente produto acetilado 4c (e.e. 82 %) em bons excessos enantioméricos. Conclui-se que os fungos de origem marinha utilizados neste trabalho são potenciais fontes de epóxido-hidrolases para promover a abertura seletiva de epóxidos. / In this work carried out itself the first study biocatalytic involving reactions of reduction of cetonas with fungi of marine origin. They were utilized 7 cetonas commercial as substratos and 8 fungi derived little seas like biocatalisadores. The fungi were isolated of the sponges little seas Geodia corticostylifera (Trichoderma sp Gc1, Penicillium miczynskii Gc5, Aspergillus sydowii Gc12) and Chelonaplysylla erect (Bionectria sp Ce5, Aspergillus sydowii Ce15, Penicillium raistrickii Ce16 and Aspergillus sydowii Ce19). The reduction 2-chloro-1-phenylethanone (1) was studied under several conditions of reaction (changes of pH, addition or absence of glucose) and the best result was with fungus P. miczynskii Gc5, therefore itself obteve an isolated performance of 60% and excess enantiomeric of 50% for the (S)- 2-chloro-1- phenylethanol (1a). The interesting one in these studies was that all of the fungi utilized in the selection with the 2-chloro-1-phenylethanone (1) presented selectivity anti- Prelog. In the literature is common obtain reduction enzymatic with selectivity Prelog. To 2-bromo-1-phenylethanone (2) was biotransformaded by the fungus A. sydowii Ce19 you correspond composed: (S)-2-bromo-1-phenylethanol (2a), (S)-2-cloro-1- phenylethanol (1a), whereas to (2c), 2-chloro-1-phenylethanone (1) and the 2- phenyloxirane (2b) were obtained by reactions not enzymatic. To 2-bromo-1-(4- bromophenyl)ethanone (3) and to 2-bromo-1-(4-nitrophenyl)ethanone (4) were entirely biodegradadas by the fungus A. sydowii Ce19. The reduction biocatalytic of the 1-(2- iodophenyl)ethanol (5) and 1-(3-iodophenyl)ethanol (6) with the fungus Trichoderma sp Gc1 supplied the 1-(2-iodophenyl)ethanol (5a) and the 1-(3-iodophenyl)ethanol (6a) with excellent excesses enantiomeric (e.e. > 99%). It stayed verified also that the fungi derived little seas for promote the reactions of reduction by biocatalysis are going to be cultivated in water of the artificial sea.

A preocupação com o desenvolvimento de técnicas menos agressivas em termos ambientais contribuindo para o desenvolvimento sustentável, levou a utilização de tecnologia enzimática e materiais biodegradáveis como uma rota alternativa. A quitosana é um polímero atóxico, biodegradável e biocompatível proveniente da desacetilação da quitina, subproduto da indústria pesqueira. Uma de suas aplicações é como suporte para a imobilização de biocatalisadores com o intuito de melhorar algumas características, como estabilidade e reutilização. A imobilização pode ocorrer por diversas técnicas, não existindo um método único que abrange todo e qualquer caso. Neste contexto, esse trabalho estudou a viabilidade da utilização de um hidrogel à base de quitosana para imobilização da lipase de Rhizopus oryzae (L036P). Para isso foi realizada a imobilização da lipase por adsorção física e por ligação covalente em hidrogel de quitosana ativado com glutaraldeido. A fim de verificar modificações químicas significativas e a perda de massa das amostras em função da temperatura, os materiais foram submetidos às técnicas de termogravimetria (TG) e espectroscopia de infravermelho com transformada de Fourier (FTIR). A massa residual foi de 30% para a enzima livre e de 45% para as enzimas imobilizadas e os espectros de FTIR comprovaram a imobilização devido a mudanças ocorridas em algumas bandas de absorção. A análise da morfologia da superfície do suporte foi realizada através de imagens obtidas por microscopia eletrônica de varredura que evidenciaram uma estrutura mais densa e menos porosa após a reticulação do hidrogel. A atividade hidrolítica da lipase imobilizada foi de 406,30 U/g para a imobilização por adsorção física; 439,82 U/g para a imobilização por ligação covalente. Os parâmetros cinéticos km e Vmáx foram determinados e não houve diferença significativa no valor de km para a ambas as lipase imobilizada já o valor de Vmáx sofreu uma queda para ambas as imobilizações indicando uma possível inibição não competitiva. A estabilidade térmica e de estocagem foram avaliadas e foi observada uma melhora na estabilidade térmica após 150 minutos e a atividade hidrolítica de todos os materiais não apresentaram perda significativa após 120 dias. Os biocatalisadores foram ainda caracterizados quanto a atividade ótima de atuação em função da temperatura e pH utilizando a técnica de planejamento de experimentos ( delineamento composto rotacional 2² com três repetições no ponto central). Os resultados encontrados mostraram que há uma variação na temperatura e pH ótimo após a imobilização, sendo encontrado valores máximos de 839,76 U/g para a lipase livre (pH 7,5 a 36°C), 574,18 U/g para a lipase imobilizada por adsorção física (pH 7,5 a 50°C) e 3572,44 U/g para a lipase imobilizada por ligação covalente (pH 8,5 60°C). A partir dos resultados obtidos, verificou-se a potencialidade da utilização de hidrogel como suporte de imobilização da lipase. / The concern with the development of less aggressive techniques for the environment contributing to sustainable development, has led to use of enzyme technology and biodegradable materials as an alternative route. Chitosan is a polymer nontoxic, biodegradable and biocompatible obtained from the deacetylation of chitin by-product of the fishing industry. One of the applications of chitosan is as a support for the immobilization of biocatalysts in order to improve certain characteristics, such as stability and reusability. Immobilization may occur for several ways, with no single method that covers every case. In this context, this study investigated the viability of using a chitosan-based hydrogel to immobilization of lipase Rhizopus oryzae (L036P). To do this was made lipase immobilization by physical adsorption and covalent linking of chitosan hydrogel activated with glutaraldehyde. In order to verify significant chemical modifications and the mass loss of samples as a function of temperature, the materials were subjected to thermogravimetric analysis (TG), and infrared spectroscopy with Fourier transform (FTIR). The residual mass was 30% for the free enzyme and 45% for immobilized enzymes and the infrared spectra confirmed the immobilization due to changes in absorption in certain bands. The analysis of the support surface morphology was performed by images obtained by scanning electron microscopy which showed a denser and less porous structure after crosslinking of the hydrogel. The hydrolytic activity of the immobilized lipase was 406.30 U / g for the immobilization by physical adsorption and 439.82 U / g for the immobilization by covalent attachment. Kinetic parameters (km and Vmáx) were determined and there was no difference difference in the amount of km for both immobilized lipase. The Vmáx value has fallen for both immobilizations indicating a possible non competitive inhibition. The thermal stability and storage were evaluated and it was observed an improvement in thermal stability after 150 minutes and the hydrolytic activity of all the materials showed no significant loss after 120 days. The biocatalysts were further characterized as the optimal activity of action as function of temperature and pH using the experimental design technique through rotational composite design with three replications 2² the center point. The results showed that there is a variation in optimal temperature and pH after immobilization being found maximum values of 839.76 U / g for the free lipase (pH 7.5 and 36 °C), 574.18 U / g for lipase immobilized by physical adsorption (pH 7.5 and 50 °C) and 3572.44 U / g to covalently immobilized lipase (pH 8.5 and 60 °C). From the results obtained, it was verified the potential use of hydrogel as lipase immobilization support. / Programa Institucional de Bolsas de Pós-Graduação - PIB-PÓS

This work describes the design, synthesis and operation of a (R,R)-(+)-hydrobenzoin-diacetic acid molecular walker attached to polyether tracks containing 2, 3 or 4 secondary alcohol footholds. A macrocycle-the walker attached to a 2-foothold track by two ester linkages-was regioselectively hydrolysed by lipase AS. The resultant seco¬-acid was recyclised using Yamaguchi chemistry, demonstrating a bipedal walker stepping off, and on to, a track. These conditions were extended to tracks containing 3 and 4 alcohol footholds and the design of a 3-foothold track that incorporated a pentaethylene glycol chain. Using an information type Brownian ratchet mechanism, ~90% of walkers stepped away from the starting position, and ~68% of walkers took 2 steps to a foothold 16 atoms away. Importantly only smaller or equal to4% of walkers were found to completely detach from the track per operation cycle.

Chang Pui Sze. / Thesis submitted in: October 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 204-227). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract (In English) --- p.ii / Abstract (In Chinese) --- p.iv / List of Tables --- p.vi / List of Figures --- p.x / Contents --- p.xii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Historical Background of Sufu --- p.1 / Chapter 1.2 --- Nutritional Benefits of Sufu --- p.2 / Chapter 1.3 --- Production Steps --- p.2 / Chapter 1.3.1 --- Preparation of Tofu --- p.5 / Chapter 1.3.2 --- Preparation of Pehtze --- p.5 / Chapter 1.3.3 --- Salting or Brining --- p.8 / Chapter 1.3.4 --- Aging --- p.9 / Chapter 1.4 --- Local Varieties of Sufu --- p.9 / Chapter 1.5 --- Other Types of Sufu Fermentation --- p.10 / Chapter 1.6 --- Biochemical Changes during Sufu Production --- p.11 / Chapter 1.6.1 --- Protein Faction --- p.12 / Chapter 1.6.2 --- Lipid Fraction --- p.14 / Chapter 1.6.3 --- Carbohydrate fraction --- p.14 / Chapter 1.7 --- Sufu Flavor --- p.14 / Chapter 1.7.1 --- "Water-soluble Peptides, Free Amino Acids and Tasty Oligopeptides" --- p.14 / Chapter 1.7.2 --- Nucleotide Contents in the Taste of Sufu --- p.15 / Chapter 1.7.3 --- Effects of Ethanol on Flavor Development --- p.15 / Chapter 1.7.4 --- Volatile Components in Sufu --- p.16 / Chapter 1.8 --- Microbiological Safety of Sufu --- p.18 / Chapter 1.9 --- Existing Problems in Sufu Production --- p.19 / Chapter 1.10 --- Acceleration of Sufu Maturation by Adding Exogenous Lipase --- p.20 / Chapter 1.10.1 --- Proteases --- p.22 / Chapter 1.10.2 --- Problems with Proteases --- p.24 / Chapter 1.10.3 --- Lipases --- p.25 / Chapter 1.10.4 --- Problems with Lipases --- p.26 / Chapter 1.11 --- Replacement of Sodium Salt in Food with Alternative Salts --- p.29 / Chapter 1.12 --- Objectives --- p.33 / Chapter 2 --- Development of Volatile Compounds in Sufu --- p.35 / Chapter 2.1 --- Introduction --- p.35 / Chapter 2.2 --- Materials and Method --- p.36 / Chapter 2.2.1 --- Sufu Production --- p.36 / Chapter 2.2.1.1 --- Preparation of Tofu --- p.36 / Chapter 2.2.1.2 --- Inoculation of Tofu --- p.37 / Chapter 2.2.1.2.1 --- The Mold Strain --- p.37 / Chapter 2.2.1.2.2 --- Spore Suspension --- p.38 / Chapter 2.2.1.2.3 --- Spore Count in Spore Suspension --- p.38 / Chapter 2.2.1.3 --- Preparation of Pehtze --- p.39 / Chapter 2.2.1.4 --- Brining and Aging --- p.41 / Chapter 2.2.2 --- Sampling of Sufu --- p.42 / Chapter 2.2.3 --- Flavor Analysis --- p.42 / Chapter 2.2.3.1 --- Simultaneous Steam Distillation-Solvent Extraction (SDE) --- p.42 / Chapter 2.2.3.2 --- Gas chromatography-mass spectrometry (GC-MS) Conditions --- p.43 / Chapter 2.2.3.3 --- Compound Identification and Quantification --- p.44 / Chapter 2.3 --- Results --- p.45 / Chapter 2.3.1 --- Evolution of Volatiles During Sufu Aging --- p.45 / Chapter 2.3.1.1 --- Esters --- p.46 / Chapter 2.3.1.2 --- Alcohols --- p.51 / Chapter 2.3.1.3 --- Aldehydes --- p.55 / Chapter 2.3.1.4 --- Ketones --- p.55 / Chapter 2.3.1.5 --- Other Nitrogen-containing Compounds --- p.59 / Chapter 2.3.1.6 --- Sulfur (S)-containing and Oxygen (O)-containing Compounds --- p.51 / Chapter 2.3.1.7 --- Pyrazines --- p.61 / Chapter 2.3.1.8 --- Miscellaneous Compounds --- p.63 / Chapter 2.3.2 --- Change in the Concentrations of Sufu Odorous Compounds with Time --- p.65 / Chapter 2.4 --- Discussion --- p.67 / Chapter 2.4.1 --- Quantitatively Important Volatile Components of Sufu --- p.67 / Chapter 2.4.2 --- Esters --- p.59 / Chapter 2.4.3 --- Alcohols --- p.72 / Chapter 2.4.3.1 --- 1-Hexanol --- p.72 / Chapter 2.4.3.2 --- Phenol and 2-Methoxyphenol --- p.74 / Chapter 2.4.4 --- Aldehydes --- p.75 / Chapter 2.4.4.1 --- Hexanal --- p.75 / Chapter 2.4.4.2 --- "(E,E)-2,4-Heptadienal" --- p.77 / Chapter 2.4.4.3 --- (E)-2-Heptenal --- p.78 / Chapter 2.4.4.4 --- Benzeneacetaldehyde --- p.79 / Chapter 2.4.5 --- Ketones --- p.80 / Chapter 2.4.5.1 --- 3-Hydroxy-2-Butanone --- p.81 / Chapter 2.4.6 --- Sulfur-Containing Compounds --- p.82 / Chapter 2.4.6.1 --- 3-(Methylthio)propanal --- p.82 / Chapter 2.4.7 --- Pentylfuran --- p.84 / Chapter 2.4.8 --- Naphthalene --- p.86 / Chapter 2.4.9 --- Contaminants and artifacts generated by A-SDE --- p.87 / Chapter 2.5 --- Conclusion --- p.92 / Chapter 3 --- Acceleration of Sufu Production with Exogenous Lipase Effect on Flavor Development --- p.95 / Chapter 3.1 --- Introduction --- p.95 / Chapter 3.2 --- Materials and Method --- p.96 / Chapter 3.2.1 --- Sufu Production --- p.96 / Chapter 3.2.2 --- The Addition of Lipases --- p.96 / Chapter 3.2.3 --- Sampling of Sufu --- p.97 / Chapter 3.2.4 --- Flavor Analysis --- p.97 / Chapter 3.2.5 --- Statistical Analysis of Sufu Flavor Compounds --- p.98 / Chapter 3.2.6 --- Proximate Analysis --- p.98 / Chapter 3.2.7 --- Freeze-Drying --- p.99 / Chapter 3.2.8 --- Statistical Analysis of Sufu Proximate Contents --- p.99 / Chapter 3.2.9 --- Sensory Evaluation of Experimental Sufu --- p.100 / Chapter 3.3 --- Results --- p.102 / Chapter 3.3.1 --- Experiment I ´ؤ Adding 0.01% (w/w) Lipase from Porcine Pancreas and Candida rugosa --- p.102 / Chapter 3.3.1.1 --- Esters --- p.104 / Chapter 3.3.1.2 --- Alcohols --- p.108 / Chapter 3.3.1.3 --- Aldehydes --- p.110 / Chapter 3.3.1.4 --- 3-Hydroxy-2-Butanone --- p.113 / Chapter 3.3.1.5 --- 3-(Methylthio)propanal --- p.114 / Chapter 3.3.1.6 --- 2-Pentylfuran --- p.115 / Chapter 3.3.1.7 --- Naphthalene --- p.115 / Chapter 3.3.2 --- Experiment II - Adding 0.02% Lipase from Porcine Pancreas and Candida rugosa --- p.117 / Chapter 3.3.2.1 --- Esters --- p.118 / Chapter 3.3.2.2 --- Alcohols --- p.123 / Chapter 3.3.2.3 --- Aldehydes --- p.125 / Chapter 3.3.2.4 --- 3-Hydroxy-2 -Butanone --- p.129 / Chapter 3.3.2.5 --- 3-(Methylthio)propanal --- p.129 / Chapter 3.3.2.6 --- 2-Pentylfuran --- p.131 / Chapter 3.3.2.7 --- Naphthalene --- p.131 / Chapter 3.3.3 --- Sensory Evaluation of Lipase-treated Sufu --- p.132 / Chapter 3.3.4 --- Proximate Composition of Sufu at Different Ages from the 3 Treatments --- p.134 / Chapter 3.3.4.1 --- Addition of 0.01%(w/w) Lipase to Sufu Aging Solution --- p.134 / Chapter 3.3.4.1.1 --- Crude Protein --- p.134 / Chapter 3.3.4.1.2 --- Crude Lipid --- p.135 / Chapter 3.3.4.1.3 --- Moisture --- p.138 / Chapter 3.3.4.1.4 --- Ash --- p.138 / Chapter 3.3.4.2 --- Addition of 0.02% (w/w) Lipase to Sufu Aging Solution --- p.141 / Chapter 3.3.4.2.1 --- Crude Protein --- p.141 / Chapter 3.3.4.2.2 --- Crude Lipid --- p.141 / Chapter 3.3.4.2.3 --- Moisture --- p.144 / Chapter 3.3.4.2.4 --- Ash --- p.144 / Chapter 3.4 --- Discussion --- p.147 / Chapter 3.4.1 --- Adding 0.01 % Lipase from Porcine Pancreas and Candida rugosa --- p.147 / Chapter 3.4.1.1 --- Comparison of Total Odorous Content --- p.147 / Chapter 3.4.1.2 --- Sensory Evaluation of Experimental Sufu --- p.147 / Chapter 3.4.2 --- Adding 0.02% Lipase from Porcine Pancreas and Candida rugosai --- p.150 / Chapter 3.4.2.1 --- Comparison of Total Odorous Content --- p.150 / Chapter 3.4.2.2 --- Sensory Evaluation of Experimental Sufu --- p.151 / Chapter 3.4.3 --- Summary of Sensory and TOC Results of Lipase Experiments --- p.153 / Chapter 3.4.4 --- Impact of Lipase Addition on Different Odorous Volatile Compounds --- p.153 / Chapter 3.4.4.1 --- Esters --- p.154 / Chapter 3.4.4.2 --- Alcohols --- p.155 / Chapter 3.4.4.3 --- Aldehydes --- p.157 / Chapter 3.4.4.4 --- 3-Hydroxy-2-Butanone --- p.159 / Chapter 3.4.4.5 --- 3-(Methylthio)propanal --- p.160 / Chapter 3.4.4.6 --- 2-Pentylfuran and Naphthalene --- p.161 / Chapter 3.4.5 --- Effect of Aging on Chemical Composition of Sufu --- p.161 / Chapter 3.4.5.1 --- Crude Protein --- p.161 / Chapter 3.4.5.2 --- Crude Lipid --- p.162 / Chapter 3.4.5.3 --- Ash --- p.163 / Chapter 3.4.6 --- Effect of Lipase Addition on Chemical Composition of Sufu --- p.163 / Chapter 3.4.7 --- Effect of Lipase Addition on Free Fatty Acid (FFA) Profiles --- p.164 / Chapter 3.4.8 --- Generation of Different Classes of Esters from Animal and Fungal Lipases --- p.168 / Chapter 3.5 --- Conclusion --- p.171 / Chapter 4 --- "Partial Substitution of Sodium Chloride with Potassium Chloride in Sufu Aging Solution - Effect on Proteolysis, Bacterial Growth and Flavor" --- p.174 / Chapter 4.1 --- Introduction --- p.174 / Chapter 4.2 --- Materials and Method --- p.175 / Chapter 4.2.1 --- Sufu Production --- p.175 / Chapter 4.2.2 --- Partial Substitution of NaCl with KC1 --- p.176 / Chapter 4.2.3 --- Sampling of Sufu --- p.176 / Chapter 4.2.4 --- Bacterial Count --- p.176 / Chapter 4.2.5 --- Total and Amino Nitrogen Contents in Sufu --- p.177 / Chapter 4.2.6 --- Sensory Evaluation of Experimental Sufu --- p.177 / Chapter 4.3 --- Results --- p.179 / Chapter 4.3.1 --- Microbial Growth --- p.179 / Chapter 4.3.2 --- Proteolysis --- p.181 / Chapter 4.3.2.1 --- Total Nitrogen Content (TN) --- p.181 / Chapter 4.3.2.2 --- Amino Nitrogen Content (AN) --- p.184 / Chapter 4.3.3 --- Sensory Evalutaion of Control and KCl-Substituted Sufu --- p.186 / Chapter 4.4 --- Discussion --- p.187 / Chapter 4.4.1 --- Microbial Growth --- p.187 / Chapter 4.4.2 --- Proteolysis --- p.189 / Chapter 4.4.3 --- Sensory Tests --- p.194 / Chapter 4.5 --- Conclusion --- p.198 / Chapter 5 --- Overal Conclusion --- p.199 / References --- p.204 / Appendix I --- p.228 / Appendix II --- p.229

O objetivo deste projeto foi estabelecer um processo de síntese enzimática de biodiesel empregando sebo bovino como matéria-prima lipídica. Para o desenvolvimento deste projeto de mestrado, o trabalho experimental foi direcionado para as seguintes atividades: 1) Determinação das propriedades físico-químicas da matéria-prima; 2) Testes de seleção do derivado imobilizado mais efetivo para mediar a síntese de biodiesel a partir do sebo bovino; 3) Estabelecimento de metodologias para analisar o produto transesterificado por diferentes técnicas; 4) Otimização da síntese de biodiesel por planejamento experimental; 5) Aumento de escala e comprovação do modelo estatístico e 6) Caracterização do produto formado e comparação com o biodiesel comercial. Os resultados das análises de composição da matéria-prima indicaram que a amostra de sebo bovino atende ao padrão exigido para ser utilizado na reação de transesterificação (baixo teor de água e índice de acidez). Para os testes de triagem do biocatalisador, diferentes fontes de lipase (EC 3.1.1.3) foram imobilizadas no suporte híbrido POS-PVA e utilizadas para mediar a reação de transesterificação do sebo bovino e etanol em meio isento de solventes. Todas as reações foram realizadas nas mesmas condições operacionais (temperatura de 45°C, razão molar de 1:9 (gordura/álcool) e 400 unidades de atividade enzimática por grama de sebo bovino). Os rendimentos de transesterificação, bem como os valores de produtividade, foram os parâmetros relevantes na escolha do biocatalisador mais efetivo. Os produtos transesterificados obtidos com rendimentos superiores a 90% foram ainda submetidos a análises complementares, tais como viscosidade cinemática, espectrometria de absorção na região do infravermelho e termogravimetria. O derivado imobilizado selecionado (Pseudomonas cepacia) foi caracterizado quanto às suas propriedades bioquímicas, cinéticas e de estabilidade térmica. Um planejamento experimental foi adotado para determinar a influência do pH e da temperatura na atividade enzimática. Para o estudo da cinética enzimática foram realizados experimentos com diferentes concentrações de substrato (azeite de oliva) visando determinar os parâmetros Km e Vmax na cinética de Michaelis-Menten. Um estudo da estabilidade térmica da lipase livre e imobilizada foi realizado a 60°C para determinar a constante de desativação térmica. Na seqüência, o derivado imobilizado selecionado foi utilizado para otimizar as variáveis do processo (temperatura e razão molar) empregando a metodologia de superfície de resposta, obtendo o seguinte modelo matemático para o rendimento de transesterificação: Y = 86,89-7,46 x1-2,04 x2 em que x1 e x2 são os valores codificados para as variáveis temperatura e razão molar, respectivamente. Com os resultados obtidos, as condições ótimas de reação foram determinadas por software (T= 48ºC e razão molar 1:7 (sebo:etanol)) e então um experimento de comprovação do modelo foi realizado usando uma massa de 110 gramas de meio reacional. Os valores de rendimento da reação apresentaram uma boa correlação com os resultados preditos pelo modelo (91,62% em 8h de reação). Finalmente o produto obtido foi submetido a uma sequência de testes e análises para verificar o potencial do processo enzimático. Os testes indicaram que o processo enzimático é capaz de produzir biodiesel com boa qualidade, apesar de não atender plenamente as normas estabelecidas pela Agência Nacional de Petróleo para uso de combustíveis no país. / The objective of this project was to establish a process for enzymatic synthesis of biodiesel using beef tallow and ethanol as feedstock. For the development of this project, the experimental work was directed to the following activities: 1) Determination of the physicochemical properties of the raw material; 2) Tests for selection of the most eficiente immobilized derivative to mediate the biodiesel synthesis from beef tallow; 3) Methodology establishment for analyzing the product transesterificated by different techniques; 4) Optimization the synthesis of biodiesel by factorial design; 5) Mathematical model comprovation and increase the reaction mass 6) Characterization of the product formed and comparison with the industrial biodiesel. The results of analysis of composition of the raw materials indicated that the sample of beef tallow meets the standard required to be used in the transesterification reaction (low water content and acidity). For screening tests of biocatalysts, different sources of lipase (EC 3.1.1.3) were immobilized on POS-PVA support and used to mediate the transesterification of beef tallow and ethanol in solvent free medium. All reactions were performed under the same operating conditions (temperature of 45°C, molar ratio of 1:9 (fat/ alcohol) and 400 units of enzyme activity per gram of beef tallow). The transesterification yields and the productivity values were important parameters in choosing the most effective biocatalysts. Transesterificated products obtained with yields higher than 90% were subjected to additional tests, such as kinematic viscosity, infrared spectroscopy, thermogravimetry and 1H NMR. The immobilized derivative selected (Pseudomonas cepacia) was characterized according to biochemical and kinetics properties and thermal stability. An experimental design was adopted to determine the influence of pH and temperature on enzyme activity. To study the enzyme kinetics experiments were performed with different concentrations of substrate (olive oil) to determine the parameters Km and Vmax in the Michaelis-Menten kinetics. A study of thermal stability of free and immobilized lipase was performed at 60 °C to determine the constant of thermal deactivation. Following this, the chosen immobilized derivative was used to optimize the transesterification reaction (temperature and molar ratio) via response surface methodology, obtaining the following mathematical model (Y=86.89-7.46x1-2.04x2) for the transesterification yield, where x1 and x2 are the coded values for the variables temperature and molar ratio, respectively. Optima reaction conditions were determined by software (T = 48 ° C and molar ratio of 1:7 (tallow: ethanol)) and then a trial to confirm the mathematical model was performed using 110 g of reaction medium. The yield value showed good correlation with results predicted by the model (91.62% in 8 h reaction). Finally, the product was submitted to a sequence of tests and analysis to verify the potential of the enzymatic process. The tests indicated that the enzymatic process allows producing biodiesel with good quality, although the specifications recommended by the Brazilian Petroleum Agency (ANP) to be used as biofuel were not fully attained.

O objetivo deste projeto foi estabelecer um processo de síntese enzimática de biodiesel empregando sebo bovino como matéria-prima lipídica. Para o desenvolvimento deste projeto de mestrado, o trabalho experimental foi direcionado para as seguintes atividades: 1) Determinação das propriedades físico-químicas da matéria-prima; 2) Testes de seleção do derivado imobilizado mais efetivo para mediar a síntese de biodiesel a partir do sebo bovino; 3) Estabelecimento de metodologias para analisar o produto transesterificado por diferentes técnicas; 4) Otimização da síntese de biodiesel por planejamento experimental; 5) Aumento de escala e comprovação do modelo estatístico e 6) Caracterização do produto formado e comparação com o biodiesel comercial. Os resultados das análises de composição da matéria-prima indicaram que a amostra de sebo bovino atende ao padrão exigido para ser utilizado na reação de transesterificação (baixo teor de água e índice de acidez). Para os testes de triagem do biocatalisador, diferentes fontes de lipase (EC 3.1.1.3) foram imobilizadas no suporte híbrido POS-PVA e utilizadas para mediar a reação de transesterificação do sebo bovino e etanol em meio isento de solventes. Todas as reações foram realizadas nas mesmas condições operacionais (temperatura de 45°C, razão molar de 1:9 (gordura/álcool) e 400 unidades de atividade enzimática por grama de sebo bovino). Os rendimentos de transesterificação, bem como os valores de produtividade, foram os parâmetros relevantes na escolha do biocatalisador mais efetivo. Os produtos transesterificados obtidos com rendimentos superiores a 90% foram ainda submetidos a análises complementares, tais como viscosidade cinemática, espectrometria de absorção na região do infravermelho e termogravimetria. O derivado imobilizado selecionado (Pseudomonas cepacia) foi caracterizado quanto às suas propriedades bioquímicas, cinéticas e de estabilidade térmica. Um planejamento experimental foi adotado para determinar a influência do pH e da temperatura na atividade enzimática. Para o estudo da cinética enzimática foram realizados experimentos com diferentes concentrações de substrato (azeite de oliva) visando determinar os parâmetros Km e Vmax na cinética de Michaelis-Menten. Um estudo da estabilidade térmica da lipase livre e imobilizada foi realizado a 60°C para determinar a constante de desativação térmica. Na seqüência, o derivado imobilizado selecionado foi utilizado para otimizar as variáveis do processo (temperatura e razão molar) empregando a metodologia de superfície de resposta, obtendo o seguinte modelo matemático para o rendimento de transesterificação: Y = 86,89-7,46 x1-2,04 x2 em que x1 e x2 são os valores codificados para as variáveis temperatura e razão molar, respectivamente. Com os resultados obtidos, as condições ótimas de reação foram determinadas por software (T= 48ºC e razão molar 1:7 (sebo:etanol)) e então um experimento de comprovação do modelo foi realizado usando uma massa de 110 gramas de meio reacional. Os valores de rendimento da reação apresentaram uma boa correlação com os resultados preditos pelo modelo (91,62% em 8h de reação). Finalmente o produto obtido foi submetido a uma sequência de testes e análises para verificar o potencial do processo enzimático. Os testes indicaram que o processo enzimático é capaz de produzir biodiesel com boa qualidade, apesar de não atender plenamente as normas estabelecidas pela Agência Nacional de Petróleo para uso de combustíveis no país. / The objective of this project was to establish a process for enzymatic synthesis of biodiesel using beef tallow and ethanol as feedstock. For the development of this project, the experimental work was directed to the following activities: 1) Determination of the physicochemical properties of the raw material; 2) Tests for selection of the most eficiente immobilized derivative to mediate the biodiesel synthesis from beef tallow; 3) Methodology establishment for analyzing the product transesterificated by different techniques; 4) Optimization the synthesis of biodiesel by factorial design; 5) Mathematical model comprovation and increase the reaction mass 6) Characterization of the product formed and comparison with the industrial biodiesel. The results of analysis of composition of the raw materials indicated that the sample of beef tallow meets the standard required to be used in the transesterification reaction (low water content and acidity). For screening tests of biocatalysts, different sources of lipase (EC 3.1.1.3) were immobilized on POS-PVA support and used to mediate the transesterification of beef tallow and ethanol in solvent free medium. All reactions were performed under the same operating conditions (temperature of 45°C, molar ratio of 1:9 (fat/ alcohol) and 400 units of enzyme activity per gram of beef tallow). The transesterification yields and the productivity values were important parameters in choosing the most effective biocatalysts. Transesterificated products obtained with yields higher than 90% were subjected to additional tests, such as kinematic viscosity, infrared spectroscopy, thermogravimetry and 1H NMR. The immobilized derivative selected (Pseudomonas cepacia) was characterized according to biochemical and kinetics properties and thermal stability. An experimental design was adopted to determine the influence of pH and temperature on enzyme activity. To study the enzyme kinetics experiments were performed with different concentrations of substrate (olive oil) to determine the parameters Km and Vmax in the Michaelis-Menten kinetics. A study of thermal stability of free and immobilized lipase was performed at 60 °C to determine the constant of thermal deactivation. Following this, the chosen immobilized derivative was used to optimize the transesterification reaction (temperature and molar ratio) via response surface methodology, obtaining the following mathematical model (Y=86.89-7.46x1-2.04x2) for the transesterification yield, where x1 and x2 are the coded values for the variables temperature and molar ratio, respectively. Optima reaction conditions were determined by software (T = 48 ° C and molar ratio of 1:7 (tallow: ethanol)) and then a trial to confirm the mathematical model was performed using 110 g of reaction medium. The yield value showed good correlation with results predicted by the model (91.62% in 8 h reaction). Finally, the product was submitted to a sequence of tests and analysis to verify the potential of the enzymatic process. The tests indicated that the enzymatic process allows producing biodiesel with good quality, although the specifications recommended by the Brazilian Petroleum Agency (ANP) to be used as biofuel were not fully attained.

Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d<SUB>2</SUB>, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.

Cardiovascular disease is a major cause of mortality and morbidity in patients on chronic haemodialysis (HD). One main contributing factor is renal dyslipidaemia, characterized by an impaired catabolism of triglyceride (TG)-rich lipoproteins with accumulation of atherogenic remnant particles. The enzyme lipoprotein lipase (LPL) is a key molecule in the lipolysis of TG-rich lipoproteins into free fatty acids. The activity of LPL is reduced in HD-patients. This study was performed to elucidate various conditions and factors that may have an impact on LPL-related lipid metabolism. I. The functional pool of LPL is located at the vascular surface. The enzyme is released by heparin and low molecular weight heparins (LMWH) into the circulating blood and extracted and degraded by the liver. Heparin and LMWH are used for anticoagulation during HD to avoid clotting in the extracorporeal devices. This raises a concern that the LPL system may become exhausted by repeated administration of LMWH in patients on HD. In a randomized cross over designed study twenty patients on chronic HD were switched from a primed infusion of heparin to a single bolus of LMWH (tinzaparin).  The LPL activity in blood was higher on HD with LMWH at 40 minutes but lower at 180 minutes compared to HD with heparin. These values did not change during the 6-month study period. With heparin a significant TG reduction was found at 40 minutes and a significantly higher TG value at 180 and 210 minutes than at start. TG was higher during the HD-session with tinzaparin than with heparin. Our data demonstrate that repeated HD with heparin or with LMWH does not exhaust the LPL-system in the long term but does disturb the LPL system and TG metabolism during every HD session. II. In this study HD patients were compared with patients on peritoneal dialysis (PD) in a case control fashion. PD patients showed the same reaction of the LPL system to LMWH as HD patients. This confirmed that both HD and PD patients had the same, reduced, heparin-releasable LPL pool. The main difference was that in PD patients the TG continued to be cleared effectively even at 180 minutes after the bolus of LMWH injection. This may be due to a slower removal of the released LPL by the liver in PD patients.  III. In recent years, citrate (Citrasate) in the dialysate has been used in Sweden as a local anticoagulant for chronic HD. We performed a randomized cross over study that included 23 patients (16 men and 7 women) to investigate if citrate in the dialysate is safe and efficient enough as anticoagulant. The study showed that citrate anticoagulation eliminated the need of heparin or LMWH as anticoagulation for HD in half of the patients. However, individual optimization of doses of anticoagulants used together with citrate have to be made. IV. Recently angiopoietin-like proteins, ANGPTL3 and 4 have emerged as important modulators of lipid metabolism as potent inhibitors of LPL. Twenty-three patients on chronic HD and 23 healthy persons were included as case and controls to investigate the levels of these proteins in plasma of HD-patients and to evaluate if HD may alter these levels. The data showed that plasma levels of ANGPTL3 and 4 were increased in patients with kidney disease compared to controls. This may lead to inactivation of LPL. High flux-HD, but not low flux-HD, reduced the levels of ANGPTL4, while the levels of ANGPTL3 were not significantly influenced.  On HD with local citrate as anticoagulant, no LPL activity was released into plasma during dialysis in contrast to the massive release of LPL with heparin (LMWH). Citrate HD was not associated with a significant drop in plasma TG at 40 minutes, while both HD with citrate and heparin resulted in significantly increased TG levels at 180 minutes compared to the start values. Conclusions:  Citrate as a local anticoagulant during haemodialysis eliminates the need of heparin or LMWH in about half of the HD patients. Citrate does not induce release of LPL from its endothelial binding sites. We have shown that although HD with heparin causes release of the endothelial pool of LPL during each dialysis session, the basal pool is similarly low in PD patients that do not receive heparin. This indicates that the LPL pool is lowered as a consequence of the uraemia, per se. One explanation could be the increased levels of ANGPTL3 and 4. HD with high flux filters can temporarily lower the levels of ANGPTL4. Further studies are, however, needed to understand why LPL activity is low in patients with kidney disease.

Lipoprotein lipase (LPL) is a central enzyme in lipid metabolism. It is a non-covalent, homodimeric and N-glycosylated protein, which is regulated in a tissue-specific manner and is dependent on an activator protein, apolipoprotein CII. Dissociation of active LPL dimers to monomers leads to loss of activity. This was previously found to be an important event in the rapid regulation of LPL in tissues. The mechanisms involved in the processing of LPL to active dimers, as well as in LPL inactivation through monomerization, were unknown. We have investigated the folding properties of the LPL protein, in particular the requirements for LPL to attain its active quaternary structure and to remain in the native conformation. On expression of LPL in insect cells we found that most of the LPL protein was synthesized in an inactive monomeric form. By co-expression of LPL with human molecular haperones, especially with calreticulin (CRT), the activity of LPL increased greatly, both in the cells and in the media. The effect of CRT on LPL activity was not due to increased levels of the LPL protein, but was due to an increased proportion of active dimeric LPL. Co-immunoprecipitation experiments showed direct interaction between LPL and CRT supporting the idea that this ER-based molecular chaperone supports the formation of active LPL dimers. We showed that, bis-ANS, the aromatic hydrophobic probe 1,1.-bis(aniline)-4,4.- bis(naphthalene)-8,8.disulfonate, can be used to obtain specific information about the interaction of LPL with lipid substrates and with apoCII. Bis-Ans was found to be a potent inhibitor of LPL activity, but apoCII prevented the inhibition. Our results suggest that bis-Ans binds to three exposed hydrophobic sites, of which one is at or close to the binding site(s) for apoCII. In studies of the mechanisms responsible for the spontaneous inactivation of LPL, we showed that active LPL is a dynamic dimer in which the subunits rapidly exchange partners. The rapid equilibrium between dimers and monomers exists even under conditions where LPL is relatively stable. This supports the idea that the dimer is in equilibrium with dimerization-competent, possibly active monomers. This dimerization-competent intermediate was also implicated in studies of the inactivation kinetics. The inactive LPL monomer was found to have a stable, defined conformation irrespective of how it was formed. The main differences in conformation between the inactive monomer and the active dimer were located in the middle part of the LPL subunit. Experiments with bis-Ans demonstrated that more hydrophobic regions were exposed in the inactive monomer, indicating a molten globule conformation. We concluded that the middle part of the LPL subunit is most likely engaged in the formation of the active LPL dimer. The dimerization-competent LPL monomer is a hypothetical conformational state, because it has not been possible to isolate it. To study complete refolding of LPL we used fully denatured LPL and were able to demonstrate that the recovery of LPL activity was about 40% when the denaturant was diluted by a buffer containing 20% human serum and 2M NaCl. Further studies identified calcium as the component in serum that was crucial for the reactivation of LPL. The refolding of LPL was shown to involve at least two steps, of which the first one was rapid and resulted in folded, but inactive monomers. The second step, from inactive monomers to active dimers, was slow and calcium-dependent. Also inactive monomers isolated from human tissue were able to recover activity under the influence of calcium. We proposed that calcium-dependent control of LPL dimerization might be involved in the normal post-translational regulation of LPL activity. In conclusion, LPL is a relatively unstable enzyme under physiological conditions due to its noncovalent dimeric structure. The energy barrier for folding to the active dimer is high and requires the presence of calcium ions and molecular chaperones to be overcome. The dimeric arrangement is probably essential to accomplish rapid down-regulation of LPL activity according to metabolic demand, e.g. in adipose tissue on fasting.