ENZYMOLOGY

Author: AVILÉS PUIGVERT MONTSERRAT.
Year: 2003.
University: GRANADA [www.ugr.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: HOSPITAL COMARCAL DE MOTRIL.

Summary: Hypertension leads to a systemic vascular impairment involving different target organs, including the kidney and retina. The hypertensive nephropathy is a nefroangioesclerosis with sphincter injuries in the afferent and efferent renal arterioles. In hypertensive retinopathy affects the retinal microvasculature manifest with different clinical manifestations depending on the characteristics of hypertension (format, age, control, years of evolution, â |), their effects on the underlying vascular bed, and the way etiopatogénica of injury on it. There are multiple serum and urinary biochemical markers of renal function is usually determined, and others, unusual detected in the urine, now regarded as a marker of early renal damage, as is the N-Acetil-Beta-Glucosaminidasa, which is the basis of our study. At the same objectives, in addition to studying the classical parameters of renal function and urinary activity of the NAG was looking for the correlation between them, all in relation to the degree of hypertensive retinopathy. We found no correlation between the degree we wanted affectation retinopathy and urinary excretion of NAG, considering that the production mechanism of injury in different organs may be different and not match the estadió evolutionary (damage to the renal vasculature more with respect to early retinal).

Author: ARANAZ CORRAL INMACULADA.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CC. QUÍMICAS.
Place of preparation: INSTITUTO DE ESTUDIOS BIOFUNCIONALES DE LA UCM.

Summary: This thesis has obtained a biocatalizador immobilized from a cellular extract from Agrobacterium radiobacter for the synthesis of a D-aminoácido of industrial interest, p-hidroxifenilglicina (p-HPG). The extract contains two cellular enzyme activities, D-hidantoinasa and D-carboamilasa, which transformed the substrate of departure, p-hidroxifenil-hidantoina, in the final product, p-hidroxifenilglicina. As media for restraint, have been used polymers of natural origin; chitin and quitosano obtained from waste from the fishing industry and alginate commercial origin. We have experimented with three methods of restraint, restraint coavalente, adsorption and encapsulation. In the first two, has studied the effect of the physicochemical characteristics of chitin and quitosano. In the third method, have been prepared complexes polielectrolitos of alginato-quitosano in which it has studied the effect of the method of preparation and the characteristics of quitosano employee (molecular weight and degree of acetylation). In all the methods tested, were obtained derivatives presenting both enzymatic activities and therefore valid for the synthesis of p-HPG. In all cases, derivatives are optimized by changing variables of the reaction, size medium. Derivatives optimziados have been characterized with respect to the effect of temperature and pH in their activity and stability, reuse and stability during storage. Of the carriers studied, the chitin has given better returns in the quitosano both covalent immobilisation as in the detention by adsorption. Of the quitinas studied obtained from the shell of prawns (Ploeticus mà ¼ lleri) and shrimp (Penaeus caramote) have submitted yields higher. The derivatives obtained by adsorption or enalce are not recommended for use as occurs in the first desorption of proteins immobilized with time and the latter's reaction detention affects a residue catalytic and thus gets poor performance. Derivatives encapsulated give the best returns and is also possible to reuse the biocatalizador.

Author: BAYES PUIG ÁLEX.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: Study of the properties exnimológicas of smetalocarboxipeptidasas digestive tract of lepidopteran. Especially has characterized the procarboxipeptidasa to Helicoverpa armgera i of the procarboxipeptidasa B Helicoverpa ze., Both from a structural point of view i functional. In this work are characterized for the first time canbios level of three-dimensional structure of proteins that make them resistant to specific inhibitors.

Author: Gandía Herrero Fernando.
Year: 2004.
University: MURCIA [www.um.es].
Place of defense: Facultad de Biología. Universidad de Murcia.
Place of preparation: Facultad de Biología.

Summary: This Doctoral Thesis, indicating doctorate European reflects the work done in the characterization of the pigment plant betalaínas and in the elucidation of the routes biosintéticas leading to its formation. It describes new reactions in the metabolism of these water-soluble pigments interest food, and propose a new scheme biosintético. It highlights the relevance of tirosinasa enzyme, which is capable of catalyzing with high affinity hydroxylation of betaxantinas monofenólicas. Likewise, it describes the transformation of betaxantinas difenólicas in ways leuko similar to betacyanin betanidina, which also is described as substrate tirosinasa. One of the most intriguing features of this enzyme is their ability to exist in an inactive or dormant. Tirosinasa be activated by a variety of agents, including the induction of changes in its structure by the presence of dodecil anionic detergent sodium sulfate (SDS) and partial proteolysis. In this sense, it describes the presence of latent forms of tirosinasa a source formatter of betalaínas and describes in detail the phenomenon of activation. This leads to demonstrate the existence of a common mechanism in the activation mediated by detergents and proteases and allows the development of new kinetic models. The characterization of the activity tirosinasa on various betalaínas is based on the development of protocols for the synthesis, extraction and purification of these pigments. This leads also to study their properties fisiquicoquímicas, demonstrating the existence of fluorescence visible in betaxantinas. All species analyzed show a similar pattern, with maximum excitation between 463 and 475 nm and emission peak between 506 and 515 nm. Based on these properties is developing a new method for analyzing betaxantinas in complex mixtures by fluorescence detection and used a filtering system to observe the contribution of fluorescence in the structures that contain them. In comparative analysis of the spectra of bataxantinas and betacyanins reveals a solpamiento between the emission spectrum of the first and last absorbance delas. This situation results when both types of pigments are together, the reduction of fluorescence in a process of physiological effect of internal filter.

Author: WILSON SOTO LORENA.
Year: 2004.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE CIENCIAS. DEPARTAMENTO DE BIOLOGÍA MOLECULA.
Place of preparation: INSTITUTO DE CATALISIS Y PETROLEOQUIMICA. CSIC..

Summary: In this work we have prepared aggregates entrecruzados enzyme (CLEAs) of different enzymes, in order to (1) increase the enzyme stability, (2) stabilization structures cuaternarias, (3) prepare CLEAs encapsulated in Gels polyvinyl alcohol (PVA) and (4) develop a new type of biocatalizador, called CLEA with microenvironments. This new biocatalizador combines the good properties of CLEA with hydrophilic properties of a microenvironment, boosting the stability of enzymes in the presence of a cosolvent and modulate their catalytic properties. Were prepared CLEAs of penicillin G acilasa (CPA), achieving obtain a catalyst 700UI / g specific activity that is a very high value compared with other derivatives of the enzyme preparations on a solid support. These CLEAs presentap greater thermal stability that the enzyme soluble and immobilized enzyme on support Eupergit. These derivatives also helped stabilize the complex structures of enzymes multiméricas, with a very simple preparation protocol. Were prepared CLEAs two catalasas (enzymes tetraméricas) and noted the stabilization of the structure of the two enzymes were also these derivatives have greater thermal stability that the soluble enzyme and those inmovilizads in support glioxil Agarose. Encapsulation of CLEAs of PGA in more rigid polymer matrix composites alcohol polivinilico (LentiKats ®), yielded a new catalyst named LK-CLEA with better mechanical properties compared with CLEAs. Moreover, these LK-CLEA show a high stability in the presence of a cosolvent (LK-CLEA maintains el1 00% of its business while the CLEA has lost 50% of its initial activity, after 9 hours in 75% (v / v ) dioxano). These results are consistent with the proposed mechanism Exclusion codisolvente from el'LentiKats, which was also confirmed by studies partition dioxano between the external environment and LentiKats. The LK-CLEA are particularly useful for the synthesis of antibiotics (1-lactámicos (Cefalosporina G) in high concentrations codisolvente, reaching higher yields synthesis thermodynamics and cinéticamente controlled, compared with CLEAs without encapsulate; this could be due to a partition substrate (more hidrofilicos) to the interior of LentiKats and products (hydrophobic) outside it. was prepared a new CLEA, called CLEA with microenvironments (CLEA-OP), in which the soluble enzyme was precipitated in the presence of polietilenimina (P) and dextran sulphate (O). Preparation of these new derivatives considered studying molecular polymers with different masses and different proportions polímeros-enzima. CLEA-OP provided greater stability in the midst organic (t1 / 2230 h; 4 ° C 75% (v / v) dioxano) that the PGA immobilized in support Glioxil agarose (t1 / 2 47.5 h) however the thermal stability of CLEA-OP was lower (50 ° C in aqueous medium), which leads to the conclusion that the stability of CLEA-OP amid organic is due to the hydrophilic microenvironment surrounding the enzyme rather than a rigidificación catalyst. This was confirmed by experiments that showed a preferential solvatación hydration of CLEAs-OP. These catalysts microenvironment with a highly hydrophilic allowed to obtain high yields of thermodynamically controlled synthesis of cephalosporin G (97%) and enhance the behavior of the enzyme reactions in the synthesis cinéticamente controlled high concentrations of dioxano was finally prepared 8 CLEAs d 44d and lipase from Alcaligenes sp. (CLEA-QL) and Candida antarctica B (CLEA-CAB) with different microenvironments (PyO) And these catalysts were evaluated in hydrolysis reactions of chiral substrates to assess modulation of the catalytic properties depending on the microenvironment of catalyst . significant changes were observed in the enantioselectividad of the enzyme depending on the microenvironment of which comprised the CLEA. For example, the CLEA-QL in the hydrolysis of (R, S) butyrate glycidol, changed its enantioselectividad from 4 to 14, 2. addition, we noted also that these CLEAs with microenvironments lipase had a high stability in the presence of a cosolvent.

Author: MOLDES MOREIRA DIEGO.
Year: 2004.
University: VIGO [www.uvigo.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: DPTO. DE INGENIERÍA QUÍMICA UNIVERSIDAD DE VIGO.

Summary: There have been enzymes ligninolíticos manganese peroxidase and laccase, through the cultivation of putrefaction white fungus Phanerochaete chrysosporium and trametes versicolor. The culture media usually include lignocellulosic waste that allow an induction of the enzyme production. The enzymes produced in this way were used as agents of degradation of dyes, which because of its variety and similarity to many xenobiotics compounds may be considered polluting model. Thus studies bleaching permitén information on the ability of degradation constaminantes by enzymes ligninolíticos. With phanerochaete chysosporium studies were conducted bleaching in vivo and in vitro. In studies of co-existing bleaching processes in vivo enzymatic degradation and adsorption. By bleaching in vitro was to develop a method of docoloración with additions of manganese peroxidase and hydrogen peroxide to improve the results of discoloration, as well as an ongoing treatment in time. In different studies of the enzyme laccase discoloration of Trametes vesicolor concluded that the way to producicón of this enzyme about influences on the ability of discoloration extracellular fluid produced. The differences are due to factors obtained enzymatic and non-enzymatic. The enzyme factor refers to the low molecular weight substances produced in crops capable of producing bleaching of certain structures themselves. This activity is mediated paor the presence of peroxides produced by the fungi themselves. The enzyme factor is given by the difference in composition of the enzyme complex produced, in particular, the difference in the proportion of laccase isozymes present in each type of crop. It reached this conclusion after carrying out studies identifying the isoenzymes produced, purification, and quantification of the same, as well as subsequent uan relationship between the proporcióin of isoenzymes and the ability to bleaching. We have also used the purified isozymes of laccase to conduct studies with bleaching systems lacasa- mediator, thanks to the qu laccase degradation occurs faster and even expand the range of products oxidables. The application of various mediators resulted muyn effective in terms of speed of discoloration, as well as the grdo bleaching observed. In mediation capacity was observed for the following order, for mediators employees: PZ HBT MORE NNDS MORE MORE MORE ApHB. Adding d eun mediator in a reaction with laccase degradation can also modify the mechanism of reaction occurred. Thus, the polymer produces phenol red dye in its reaction with laccase, but if you add the mediator HBT, the system laccase -HBT get a further degradation of phenol red without producing this type of compound. Finally, we examined the effect of biocide pentachlorophenol in crops Trametes versicolor. This produces a compound induction of extracellular laccase activity, the unemployment effect also occurs at the level of gene transcription, as shown by RT-PCR technique.

Author: Monegal Granados Ana.
Year: 2004.
University: RAMÓN LLULL [www.url.edu].
Place of defense: Escuela Técnica Superior IQS.
Place of preparation: Escuela Técnica Superior IQS.

Summary: The glicosiltransferasas are enzymes responsible for the biosynthesis of glycoconjugates. Its implications for biomedicine has become very attractive targets for studies estructura-función. The -1.3 -galactosiltransferasa, the subject of this thesis, is responsible for the synthesis of disaccharide terminal Gal 1.3 Gal-, antigen majority in the xenotransplante. At present, the availability of human organs for transplantation is a serious problem given the increasing demand for organs and the shortage of donors. The xenotransplante or organ transplant animals to humans, it could be a temporary solution to this problem. However, the biggest drawback of xenotransplante is the rejection hiperagudo caused by the immune system response to antigen-specific animal tissues. The antigen is epítopo Gal majority or xenoantígeno, which is abundantly expressed on the surface of endothelial cells of all mammals except humans and apes in the old world. The epítopo Gal is produced by the enzyme -1.3 -galactosiltransferasa (3GT). Various strategies have been proposed to prevent rejection hiperagudo: a) genetic manipulation of the donor animals (pigs), to produce organs that do not express the epítopo b) neutralizing antibodies natural human synthetic structures containing epítopo c ) elimination of antibodies after transplantation, d) lock acceptor substrate by overexpression of other glicosiltransferasas in the donor animal, and e) inhibition of enzyme activity 3GT in tissue to transplant. The objective of this work is the molecular characterization of the 3GT bovine. The research focuses on the study estructura-función its catalytic mechanism, using protein engineering techniques and enzymology. This has clonó the catalytic domain of the 3GT and expressed in E.coli. It was developed and validated different tests to characterize the enzyme activity wt. Then, according to the crystallographic structure of the protein was analyzed in different positions mutacional selected as candidates to participate in catalysis or binding to the substrates. These mutants were characterized cinéticamente and one of them, who was inactive, was studied in depth by subjecting it to chemical experiments rescue to recover its activity.

Author: PARDO HONRUBIA ALEJANDRO.
Year: 2004.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: INSTITUTO DE CATALISIS Y PETROLEOQUIMICA.
Place of preparation: INSTITUTO DE CATALISIS Y PETROQUIMICA (CSIC).

Summary: Introduction. Currently interest in the H2 as a vector of energy is very large due to increased CO2 in the atmosphere produced by the use of fossil fuels and their potential impact on climate change. Since millions of years ago many microorganisms generate energy metabolism by oxidation of H2. The living beings exploit their energy resources through sophisticated catalysts: enzymes. The hidrogenasas are enzymes that catalyze the reversible oxidation of the molecule H2. The high efficiency of these catalysts is used by many microorganisms in nature to obtain energy through metabolic pathways, or to remove excess protons and electrons [R. Cammack, 2001]. From the technological point of view, hidrogenasas have application in the development of production reactors H2 and fuel cells [SPJ Albracht, 1994; R. Cammack, 2001; R. Mertens Et al., 2004], and the structural and functional knowledge of hidrogenasas is essential for the progress of these applications. On the other hand, computational chemistry (Molecular Modeling) is a software application for the simulation of chemical processes and characterization of chemical compounds through molecular models. Currently, it is a very powerful tool in the service of chemistry pilot either to complement and support experimental results, or in other cases to obtain experimental results that chemistry is not able to obtain or can hardly do so by problems of complexity, cost, time limitation technological or dangerous experiments. In carrying out part of this thesis, it has been necessary to use computational methods that are plausible and the need to address these issues through methods that the experimental techniques by themselves have been unable to respond in a clear way so far. Objectives. The fundamental objective has led to the realization of this thesis has been to deepen the knowledge electrónico-estructural and functional of hidrogenasas of NiFe. To achieve this goal, and presented in light of the introduction of what is known and remains to be known about this type of hidrogenasas, have raised a number of more specific objectives that have touched various aspects of the survey of hidrogenasas of NiFe: -Caracterización structural different states of redox active detected espectroscópicamente of hidrogenasas of NiFe. Characterization-electronic states of oxidation of Fe and Ni in the forms detected espectroscópicamente -Caracterización the functionality of the active center in the breakdown of the molecule H2. - Characterization of proton transport between the active and environment of the protein. - Functional characterization of the activation and inactivation processes. Together these objectives partial proposes a fairly complete picture of what is happening in the hidrogenasas of NiFe during the catalytic activity, specifically in the active center and its environs, in order to contribute to the advancement and better understanding of these hidrogenasas. Conclusions. The final key findings obtained from the results and discussions carried out in this thesis are summarized in the following points: "The state of oxidation of Ni in ways undetectable in EPR is a state Ni (II) high spin." Mechanism rupture of the molecule H2 occurs through a mechanism rupture heterolítico, with the participation of the Faith as a reception center of the molecule H2. Moreover, this process is favored when the active center is capable of oxidized by loss of an electron. "The proton transfer between the active and environment of the protein involves the participation of glutamic acid (Glu-25 in D. fructosovorans, and Glu-18 in D. gigas) in the processes of production and consumption of H2." It is proposed that the difference between the ways Ni-Ay Ni-B is that the former presents or 8 n (OH-4a0) and a group sulfenato (S = O), while the second contains only one (OH), being phase limiting activation process of how Ni-A conversion of the groups (OH-) and (S = O) to a group (O2H-); needed másresultados pilot to confirm this hypothesis. Probably the limiting step in the process of inactivation of how Ni-B is due to the stabilization of a molecule of H2O by glutamic acid (Glu-25 in D. fructosovorans, and Glu-18 in D. gigas), which should come into the center to give rise to active (OH).

Author: SOSA AZUL M. JOSÉ.
Year: 2004.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: In the present paper has studied the evolution of the microbial population and different physico-chemical parameters s (humidity, water activity and chloride) in the interior of cured ham Check these parameters are not affected by the factor of the level of FAT perniles. Inside the cured ham is developing a microbial population that is maintained throughout the trial, being primarily consists micrococáceas, yeast and lactobacilli. We have isolated microorganisms along the processing hams and has been studied their enzymatic activities. Thus, the microbial population isolated during the early stages of processing has increased proteolytic activity and lipolítica, coinciding with a reduced availability of soluble nitrogen compounds and free fatty acids in ham. The activity aminasa of microorganisms evaluated is essentially cell type, generally little selective against the various amino acids, and higher in the drier and isolates cellar, where there is an increased presence of free amino acids. We have selected a group of microorganisms isolated from the interior of cured ham for possible use as initiators crops in parts Meat mature high-volume, as a cured ham. The selection was made in accordance with their enzyme activity in vitro for their post in a model system meat matured. It was noted that these microorganisms are properly presented and proteolytic activity and lipolítica exceed the residual muscle activity. These enzymatic activities are dependent on the type of microorganism. The presence of microorganisms isolated from the interior of cured ham on meat matured, also caused an increase in the variety and concentration of volatile compounds detected during ripening, especially those related to the catabolism of amino acids, which do not have a large impact in the sensory properties of meat products matured. In response to the generation of precursor chemicals and volatile organic compounds by different isolates meat matured, and their potential complementarity, have proposed some isolated (Staphylococcus xylosus Sxf01, Lactobacillus brevis Lbc05 and Debaryomyces hansenii Dhc01) for its use as initiators crops in cured ham .

Author: Sidrach de Cardona Paniagua Lara.
Year: 2005.
University: MURCIA [www.um.es].
Place of defense: Facultad de Química.
Place of preparation: Facultad de Química.

Summary: It has been dealt with the purification and characterization of kinetic proteases, peroxidases and polifenoloxidasas obtained from agro-industrial waste artichoke. The purification process has covered enzymes stages centrifuge, chromatographies ultrafiltration and ion exchange, hydrophobic interaction and gel filtration, considering its escalated to the level of preparation pilot plant. The progress of the process has been tested by PAGE (SDS) and IEF, techniques have also permitidola determination of molecular masses and pH values of isoelectric purified isozymes, with satisfactory recovery and degree of purification. Based on the pistils of artichoke, have obtained three aspartic proteases or Cinarasas A, B and C. The first is the most abundant and the second presents the highest specific activity. They show activity amidolítica and inhibition before pepstatina, intermediate between pepsin and quimosina. The clotting activity of milk and proteolytic activity on casein, is similar to that of cuajos veal and microbial, which may have application in developing cuajadas and cheeses. Bracts artichoke contain cationic peroxidase in states penta and hexacoordinados with different proportions of calcium linked. It has characterized the activity and stability of this peroxidase before calcium, hydrogen peroxide and organic solvents, optimizing the test conditions for their potential applications of biotechnology, such as clinical laboratory tests (glucose, cholesterol, immunoassays for viruses, etc.). Degradation phenolic pollutants and synthesis of phenolic resins. Also from bracts artichoke, it has been purified tirosinasa or polifenoloxidasa, characterizing their activities monofenolasa and difenolasa. The enzyme acts well in aqueous medium, and to organic surfactants, showing estéreoselectividad. Trials of activity and stability have led to optimal reaction conditions for the development of subsequent studies on its potential biotechnology applications in analysis, synthesis and degradation of phenols.

Author: GALLEGO MOLI ORIOL.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: Dep.de Bioq.y Biol. Molecular, U.A.B..
Place of preparation: Universitat Autònoma de Barcelona.

Summary: For the first time, our group identified an enzyme of the superfamily of aldo-ceto reductasas (AKR), AKR1B12, active with retinoids (Crosas et al., 2001). Since AKR1B12 presented a sequence identity close to 70% with enzymes human AKR1B1 and AKR1B10, it was decided to extend the study of the activity retinoid oxidoreductasa between AKR. In this PhD thesis, we have deepened in the study of retinal reductase activity of members of the superfamily of AKR. The work began with the characterization of in vitro activity with retinoids of AKR human family AKR1. In cooperation with the group of Dr. Natalia Kedishvili (now at the Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, USA) performed a comparative analysis of the activity Retinol oxidoreductasa enzymes of superfamilias of deshidrogenasas / reductasas medium chain (MDR), deshidrogenasas / reductasas short chain (RDS) and AKR. We used a new method based on the solubilization of retinoids with BSA, and analysis by HPLC. It determined the values of the kinetic constants for human enzymes from the three superfamilias without the inhibitory effect of Tween-80, a detergent used traditionally for the kinetic study of MDR and AKR. These results showed that the value of Km for retinoids is similar (0,1-1? M) in the enzymes studied. The main differences are focused on the value of kcat, which ranged up to three orders of magnitude between the different enzymes. Another notable result was the demonstration that none of the enzyme was able to use as a substrate retinol or retinal, coupled with the cellular protein unidora retinol (CRBPI). Until the present study, using two arguments to defend a major role in the RDS in the oxidation of retinal to Retinol, the first phase synthesis of retinoic acid: 1) Securities km below the RDS for retinoids (up to two orders regarding the magnitude lower ADH). 2) Ability to recognize the holoCRBPI as substrate, a seemingly exclusive property of some RDS. Our group has ruled these two arguments and has introduced AKR as third group enzyme involved in metabolism of retinoids. The second part of the thesis focuses on the structural and functional study of the AKR1B10, AKR introducing greater efficiency catalyst for the reduction of retinal. This enzyme is sobrexpresa in different types of human cancer, which increased our interest in determining its role in the path of retinoic acid synthesis. In addition to the in vitro study of the activity retinoid oxidoreductasa of the enzyme were also carried out an in vivo study. Using cells COS-1 as a model, and by transient transfection, we were able to witness AKR1B10 also participates in the reduction of retinal in vivo, but it was not able to oxidize retinol. Finally, in collaboration with the group of Dr. crystallography. Ignasi Fita (Parc Científic de Barcelona-CSIC), was obtained three-dimensional structure of the complex AKR1B10- NADP + -tolrestat. The Tolrestat is an inhibitor of AKRB1 widely studied as a drug in the treatment of secondary complications of diabetes, but also inhibits AKR1B10 both in vitro and in vivo. Everything and the high sequence identity between AKR1B1 and AKR1B10, these enzymes present values very different kinetic constants for the reduction of retinal. Thanks to the resolution of the structure of AKR1B10, we hope to study the determinants estruct 8 urales q 36b ue give each enzyme kinetic properties characteristics, as well as setting the foundation for the design of new specific inhibitors for each of them.

Author: GARCÍA MURRIA MARÍA JESÚS.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE MATEMÁTICAS - UNIVERSITAT DE VALÈNCIA.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS - UNIVERSITAT DE VALÈNCIA.

Summary: The Ribulosa 1 ,5-bisfosfato carboxylase oxigenasa (Rubisco) catalyzes the first step in the photosynthetic fixation of C02 via the Calvin cycle. The structure of holoenzima active in eukaryotic organisms is a hexadecámetro, consisting of 8 subunits large (51-58KDa) and 8 small subunits (12-18KDa). In terms of senescence natural or induced stress the Rubisco undergoes rapid degradation and selective. One of the more generalized responses to different kinds of stress in different organisms is the oxidation of thiol groups of Rubisco, which has been linked in vitro and in vivo with the inactivation and susceptibilización proteolytic enzyme. Waste cisteinas phylogenetically conserved of Rubisco are potential candidates to mediate this redox regulation. Against this backdrop, the overall objective of this study was to evaluate the involvement of waste Cys172y Cys192 (highly conserved in eukaryotes and cyanobacteria) in the activity, modulation redox, and the structural organization of holoenzima. This would have addressed the following: I. Obtaining and characterization of Rubiscos mutants C172S, C192S and C172S/C192S: The roles of the residues Cys 172 and Cys 192 have been investigated studying the mutant phenotype in which such waste have been replaced by serine directed mutagenesis using gene rbcL in the unicellular alga Chlamydomonas reinhardtii.El enzyme C1728 has a specificity factor greater than the wild, with some apparent Michaelis constant for the C02 and 02 aumentadas.La Rubisco C1928 has a special sensitivity to inactivation by modifiers of grupossulfhidrilo. That seemed to respond to critical nature of the modification of Cys172 in enzyme activity and alefecto protective about this residue exercises Cys192.El study testing the thermal sensitivity of changes in the proteolytic sensitivity and the movilidaddel holoenzima in gels native indicates that the replacement of Cys 172 and / or Cys 192 affects the structure of the enzyme. II. Determination of the structure of Rubiscos C172S and C192S by X-ray diffraction: It has been determined the three-dimensional structure of the Rubisco of mutants C1728 and C1928. As most significant difference was detected a shift in the thread 131 per barrel a/13 in enzyme C1728 in relation to the structure of the enzyme wild. III. Study of the response of mutants with different types of stress: The mutant C 1728 subjected to salt stress degrades Rubisco so that the slower strain or losde more mutants. Furthermore, under a stress diamida specifically addressed to the oxidation of sulphydryl groups, the mutant C1728 is resistant to inactivation suffered other strains. This points to a role singulardel residue Cys172 in modulating the catabolism of Rubisco in response asituaciones of estrés.En stress conditions by diamida, all mutant strains (C1728, C1928 and C1728/C1928) show a degradation of the Rubisco by delayed compared with the wild strain. This suggests that, under these conditions direct oxidation of the thiol cell, the formation of a disulfide bridge between the Cys 172 and Cys192 could act as a serial condition of degrading enzyme.

Author: Larroy Hernando Carolina.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: Univ.Autónoma de Barcelona.
Place of preparation: Universidad Autónoma de Barcelona.

Summary: This thesis presents the identification and characterization of two new alcohol deshidrogenasas in the yeast Saccharomyces cerevisiae. There are two enzymes that belong to the macorfamilia alcohol deshidrogenasas medium chain, and specifically to the family of cinamil alcohol deshidrogenasas. These proteins homodiméricas composite of 40 kDa subunits. These enzymes, Adh6p and Adh7p, have a catalytic activity dependent NADP (H) and are able to oxidize aliphatic alcohols and linear or branched aromatic alcohols with or without substituents, their corresponding aldehydes, this being a reversible reaction. In fact, the two enzymes have a better catalytic efficiency compared to the reduction of aldehydes, can consider their role as that of aldehyde reductasas dependent NADP (H). It has been determined the crystal structure of Adh6p. The structure keeps domains characteristic of the alcohol deshidrogenasas, a structural domain and a coenzyme binding domain, each with an atom of zinc. Among both domains is the catalytic center where the substrate is placed oriented catalytic zinc atom and molecule of cofactor nicotinamide. The structure gained has allowed us to postulate that Adh6p works with a mechanism of catalytic 'half-of-the-sites', so that only one of the two subunits is active in a catalytic cycle. The study of these functional proteins has allowed us to note that Adh7p virtually no expressed in the yeast cell. However, Adh6p expressed during the logarithmic phase of growth and participates in functions detox aldehydes derived from ligninolisis, response to oxidative stress, in the formation of higher alcohols and maintaining the redox balance NADP + / NADPH intracellular.

Author: Lozada Ramírez José Daniel.
Year: 2005.
University: MURCIA [www.um.es].
Place of defense: Facultad de Biología.
Place of preparation: Facultad de Biología.

Summary: This Doctoral Thesis on the works developed cloning, overexpression and biochemical characterization of the enzyme S-adenosilhomocisteína hydrolase and the resulting synthesis product enzyme S-adenosilhomocisteína. In it, describes the development of a new method for measuring the activity, which is applicable in the search and selection of microorganisms producing the enzyme. The thesis was first reported cloning of the gene coding for the synthesis of S-adenosilhomocisteína hydrolase, from microorganisms mesofílicos and thermophilic of industrial interest. It was also reported the expression of enzymes from these sources at high levels, its purification and biochemical characterization of s-adenosilhomocisteína hidrolasas purified. The biochemical characterization has allowed to select one of the recombinant enzyme obtained in its possible application as biocatalizador in the synthesis of S-adenosilhomocisteína, which has been used in a bioreactor.

Author: GOUVEIA CARLOTO ANTONIO MANUEL.
Year: 2005.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The ADP-ribosa free is a nucleoside difosfoazúcar (NDP-azúcar), which has a character with ribose reducer and is toxic because of its ability to react in a non-enzymatic with side chains of proteins. It has recently been described is a driver of some ionic channels. The ADP-ribosa hidrolasas are potential protective agents that inhibit the intracellular accumulation of ADP-ribosa. In mammals, are known two types of enzymes with activity ADP-ribosa hydrolase: 1-ADP-ribosa pirofosfatasas highly specific low-km (less 1 uM) so dependent MG2 + hydrolyzed only ADP-ribosa and similar non-physiological IDP - ribose. 2-Nucleósido difosfoazúcar or difosfoalcohol (NDP-X) pirofosfatasas less specific, in addition to A (I) DP-ribosa hydrolyzed other substrates NDP-X not reducers. In this thesis, we describe that excerpts from human placenta contain two ADP-ribosa hidrolasas, which were characterized after a purification of about 1000 times. One is a ADP-azúcar pirofosfatasa: hidrolizó ADP-ribosa, ADP-glucosa and ADP-manosa, but no such UDP-glucosa at speeds similar. It looks like an enzyme previously studied in human erythrocytes and expressed from clones deNUDT5 human, but presents a Km for ADP-ribosa 5-20 times lower (7 uM). The other is a ADP-ribosa pirofosfatasa highly specific low Km (0.4 uM), the first enzyme of this type identified in human tissues. The recombinant enzyme human NUDT9, having studied its expression in E. coli, introduced properties similar to those of the ADP-ribosa pirofosfatasa placenta, including the low value of Km, which enabled us to establish the identities of the two enzymes. When incubated conH2O2 both the ADP-ribosa pirofosfatasa purified placenta as NUDT9 filed a parente "specificity of cation exchange." The activity of the enzyme in the presence of Mg2 + 5mM dropped about 10-20 times, but grew in proportion similar activity in the presence of Mn2 + 5mM. The phenomenon was accompanied by an elevation of kilometers from the ADP-ribosa pirofosfatasa for ADP-ribosa and seems due to the generation of an "oxidized" dela enzyme. The ADP-ribosa hidrolasas purified from placenta may correspond to the products of genes NUDT5 and NUDT9, which encode proteins belonging to the superfamily Nudix. Given its strict specificity and high finidad by ADP-ribosa, ADP-ribosa pirofosfatasa (NUDT9) may have a dominant role in preventing the accumulation of ADP-ribosa free in humans. This could also be the role of aDP-azúcar pirofosfatasa (NUDT5), as it may take the ADP-ribosa their most important substrate in vivo.

Author: CIMA MARTIN SERGIO DE.
Year: 2005.
University: LEÓN [www.unileon.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: UNIVERSIDAD DE LEON.

Author: Peñalver Jara María José.
Year: 2006.
University: MURCIA [www.um.es].
Place of defense: Facultad de Química.
Place of preparation: Universidad de Murcia.

Summary: The quantitative characterization of the isotopic effect of the solvent on the activities monofenolasa and difenolasa of tirosinasa, has led to establish the respective stages determinants of speed, in both activities. The activity monofenolasa has shown the release enzyme difenol in quantities estequiométricas with the kind oxitirosinasa with direct determinations by gas chromatography / mass spectrometry, as well as indirect evidence checking estequiometría variable in the reaction to various test conditions . We have designed experiments and numerical simulations integration of the corresponding system of differential equations to test the validity of various mechanisms of reaction alternative. It has been deduced the analytical expression of Factor Autoactivación of Speeds (RAF), kinetic parameter useful to quantify the self-catalytic activity monofenolasa of tirosinasa. Overall, it has demonstrated the validity of the reaction mechanism proposed for the enzyme tirosinasa by the research team, discarding erroneous mechanisms proposed by other authors. In addition, it has identified various kinetic constants that characterize the activities monofenolasa and difenolasa, tirosinasas various biological sources.

Author: Rodríguez Maynou Montserrat.
Year: 2006.
University: GIRONA [www.udg.es].
Place of defense: Universitat de Girona.
Place of preparation: Universitat de Girona.

Summary: This thesis has been marked route to internalize the onconasa, a ribonuclease cytotoxic. Results indicate that onconasa enters cells via dependent clatrina and complex AP-2. He then addresses the endosomas recycling and it is through this route that the protein exerts its cytotoxicity. Moreover, the results of this work show that PE5, a variant of human pancreatic ribonuclease (HP-RNasa) interacts with the importina through different wastes that are not sequential but are closer together in the three-dimensional structure of this protein. PM8 is a HP-RNasa with crystallographic structure dimérica cosntituida by intercanvio domain N-terminales. In this thesis have created the conditions to stabilize this dímero in solution and also proposes a mechanism for dimerización. Finally, we studied the pattern of digestion substrate by the HP-RNasa.

Author: Martínez Martínez Irene.
Year: 2006.
University: MURCIA [www.um.es].
Place of defense: Facultad de Biología. Universidad de Murcia.
Place of preparation: Facultad de Biología.

Summary: To carry out the desacetilación of cephalosporin and its derivatives, the first Ramnogalacturonan acetyl bacterial esterase (YesT) from Bacillus subtilis, was cloned and characterized. YesT was able to act on substrates cefalosporánicos and on xilano, introducing a synergistic effect when acting in combination with -xilanasa. The primary sequence alignment with other proteins revealed that YesT is a new member of the family of SGNH hidrolasas. Presents a folding sandwich. The topological diagram of YesT highlighted the divergence of all family members SGNH hydrolase from a common ancestor. This discrepancy seems to have happened in two different topological lines, one of which its members presented additional sheets to the central leaf. In addition, a Acetyl xilan esterase from Bacillus pumilus was cloned and characterized on substrates cefalosporánicos. On the other hand, developed a colorimetric method, based on a pH indicator to evaluate the activities Acetyl xilan esterase and Cefalosporin deacetilasa used as substrates 7-ACA, CPC and xilano. The method has been validated for application in high-troughput screening, assess libraries from allowing experiments directed evolution or metagenómica. To change the position 7 of the cephalosporin, a Glycine oxidase from Bacillus subtilis (GOXB) was cloned and characterized. GOXB was not able to act on the cephalosporin, it was generated by the mutant M261Y and M261H, demonstrating the involvement of this residue in the modulation of the substrate specificity. Both mutants presented a decrease in the value of KM on all substrates analyzed, but they were not active on the cephalosporin. The mutant M261Y had a value of KM on the substrate N-etil-glicina so low that adventure a role similar to that described for the enzyme N-alquil-glicina oxidase that detects N-carboxi-metil-lisina, which is a biomarcador in complications from diabetes. In order to maximize the expression of GOXB took place cultivation in the high cell density of Escherichia coli strain Rosetta (DE3). The process is carried out by providing the source of carbon exponentially albeit limiting to avoid the accumulation of acetate. The specific speeds growth of the strain could be described and optimized cell optimal concentration to carry out the induction of the expression of GOXB. Looking for new activities to GOXB, underwent directed evolution techniques showing involvement in the activity and substrate specificity of one of the waste in the consensus sequence binding to FAD. The structural modeling shows that the mutation of the Ile by Val, it seems to cause a movement in the cofactor that allows better accommodation at the heart of active substrates cyclical, as D-prolina and D-pipecólico. A Glycine oxidase from the microorganism extremófilo Geobacillus kaustophilus (GOXK) was also cloned and characterized. This enzyme introduced a broad substrate specificity, as well as properties alcalofílicas and termofílicas. The modeling showed enormous structural similarities with GOXB. The main differences appear to lie in the interaction between the monomers. However, the most important result is that GOXK is the first glycine oxidase able to act on the cephalosporin, opening new opportunities for biotechnological this enzyme.