931 resultados para proteolytic enzyme
Resumo:
Abstract Background Identification of nontuberculous mycobacteria (NTM) based on phenotypic tests is time-consuming, labor-intensive, expensive and often provides erroneous or inconclusive results. In the molecular method referred to as PRA-hsp65, a fragment of the hsp65 gene is amplified by PCR and then analyzed by restriction digest; this rapid approach offers the promise of accurate, cost-effective species identification. The aim of this study was to determine whether species identification of NTM using PRA-hsp65 is sufficiently reliable to serve as the routine methodology in a reference laboratory. Results A total of 434 NTM isolates were obtained from 5019 cultures submitted to the Institute Adolpho Lutz, Sao Paulo Brazil, between January 2000 and January 2001. Species identification was performed for all isolates using conventional phenotypic methods and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing a 441 bp fragment of hsp65. Phenotypic evaluation and PRA-hsp65 were concordant for 321 (74%) isolates. These assignments were presumed to be correct. For the remaining 113 discordant isolates, definitive identification was based on sequencing a 441 bp fragment of hsp65. PRA-hsp65 identified 30 isolates with hsp65 alleles representing 13 previously unreported PRA-hsp65 patterns. Overall, species identification by PRA-hsp65 was significantly more accurate than by phenotype methods (392 (90.3%) vs. 338 (77.9%), respectively; p < .0001, Fisher's test). Among the 333 isolates representing the most common pathogenic species, PRA-hsp65 provided an incorrect result for only 1.2%. Conclusion PRA-hsp65 is a rapid and highly reliable method and deserves consideration by any clinical microbiology laboratory charged with performing species identification of NTM.
Resumo:
This study compares two xylanases produced by filamentous fungi such as A. niger and A. flavus using agroindustrial residues as substract and evaluated the effect of these enzymes on cellulose pulp biobleaching process. Wheat bran was the best carbon source for xylanase production by A. niger and A. flavus. The production of xylanase was 18 and 21% higher on wheat bran when we compare the xylanase production with xylan. At 50°C, the xylanase of A. niger retained over 85% activity with 2 h of incubation, and A. flavus had a half-life of more than 75 minutes. At 55°C, the xylanase produced by A. niger showed more stable than from A. flavus showing a half-life of more than 45 minutes. The xylanase activity of A. niger and A. flavus were somehow protected in the presence of glycerol 5% when compared to the control (without additives). On the biobleaching assay it was observed that the xylanase from A. flavus was more effective in comparison to A. niger. The kappa efficiency corresponded to 36.32 and 25.93, respectively. That is important to emphasize that the cellulase activity was either analyzed and significant levels were not detected, which explain why the viscosity was not significantly modified.
Resumo:
Abstract Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.
Resumo:
Abstract Background Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans. Results Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA: GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA:GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose. Conclusions Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.
Resumo:
A serological follow-up study was carried out on 27 children (1–12 years old) with visceral and/or ocular toxocariasis, after treatment with thiabendazole. A total of 159 serum samples were collected in a period ranging from 22–116 months. Enzyme-linked immunosorbent assays (IgG, IgA, and IgE ELISA) were standardized, using excretory–secretory antigens obtained from the second-stage larvae of a Toxocara canis culture. The sensitivity found for the IgG, IgA, and IgE ELISA, as determined in visceral toxocariasis patients, was 100%, 47.8%, and 78.3%, respectively. Approximately 84% of the patients presented single or multiple parasitosis, as diagnosed by stool examination, yet such variables did not appear to affect the anti-Toxocara immune response. Titers of specific IgE antibody showed a significant decrease during the first year after treatment, followed by a decrease in the IgA titers in the second year, and in the IgG titers from the fourth year onwards. Sera from all patients presented high avidity IgG antibodies, indicating that they were in the chronic phase of the disease. Moreover, 1 year after treatment, the level of leukocytes, eosinophils, and anti-A isohemagglutinin in patients decreased significantly. The present data suggest that IgE antibodies plus eosinophil counts are helpful parameters for patient followup after chemotherapy.
Resumo:
A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.
Resumo:
Aims: Angiotensin-converting enzyme (ACE) inhibitors are used in diabetic kidney disease to reduce systemic/intra-glomerular pressure. The objective of this study was to investigate whether reducing blood pressure (BP) could modulate renal glucose transporter expression, and urinary markers of diabetic nephropathy in diabetic hypertensive rats treated with ramipril or amlodipine. Main methods: Diabetes was induced in spontaneously-hypertensive rats (~210 g) by streptozotocin (50 mg/kg). Thirty days later, animals received ramipril 15 μg/kg/day (R, n =10), or amlodipine 10 mg/kg/day (A, n= 8,) or water (C, n = 10) by gavage. After 30-day treatment, body weight, glycaemia, urinary albumin and TGF-β1 (enzyme-linked immunosorbent assay) and BP (tail-cuff pressure method) were evaluated. Kidneys were removed for evaluation of renal cortex glucose transporters (Western blotting) and renal tissue ACE activity (fluorometric assay). Key findings: After treatments, body weight (p = 0.77) and glycaemia (p = 0.22) were similar among the groups. Systolic BP was similarly reduced (p < 0.001) in A and R vs. C (172.4 ± 3.2; 186.7 ± 3.7 and 202.2 ± 4.3 mm Hg; respectively). ACE activity (C: 0.903 ± 0.086; A: 0.654 ± 0.025, and R: 0.389 ± 0.057 mU/mg), albuminuria (C: 264.8 ± 15.4; A: 140.8 ± 13.5 and R: 102.8 ± 6.7 mg/24 h), and renal cortex GLUT1 content (C: 46.81 ± 4.54; A: 40.30 ± 5.39 and R: 26.89 ± 0.79 AU) decreased only in R (p < 0.001, p < 0.05 and p < 0.001; respectively). Significance:We concluded that the blockade of the renin–angiotensin systemwith ramipril reduced earlymarkers of diabetic nephropathy, a phenomenon that cannot be specifically related to decreased BP levels.
Resumo:
[EN] Many ecologically important chemical transformations in the ocean are controlled by biochemical enzyme reactions in plankton. Nitrogenase regulates the transformation of N2 to ammonium in some cyanobacteria and serves as the entryway for N2 into the ocean biosphere. Nitrate reductase controls the reduction of NO3 to NO2 and hence new production in phytoplankton. The respiratory electron transfer system in all organisms links the carbon oxidation reactions of intermediary metabolism with the reduction of oxygen in respiration. Rubisco controls the fixation of CO2 into organic matter in phytoplankton and thus is the major entry point of carbon into the oceanic biosphere. In addition to these, there are the enzymes that control CO2 production, NH4 excretion and the fluxes of phosphate. Some of these enzymes have been recognized and researched by marine scientists in the last thirty years. However, until recently the kinetic principles of enzyme control have not been exploited to formulate accurate mathematical equations of the controlling physiological expressions. Were such expressions available they would increase our power to predict the rates of chemical transformations in the extracellular environment of microbial populations whether this extracellular environment is culture media or the ocean. Here we formulate from the principles of bisubstrate enzyme kinetics, mathematical expressions for the processes of NO3 reduction, O2 consumption, N2 fixation, total nitrogen uptake.
Resumo:
The corpus luteum (CL) lifespan is characterized by a rapid growth, differentiation and controlled regression of the luteal tissue, accompanied by an intense angiogenesis and angioregression. Indeed, the CL is one of the most highly vascularised tissue in the body with a proliferation rate of the endothelial cells 4- to 20-fold more intense than in some of the most malignant human tumours. This angiogenic process should be rigorously controlled to allow the repeated opportunities of fertilization. After a first period of rapid growth, the tissue becomes stably organized and prepares itself to switch to the phenotype required for its next apoptotic regression. In pregnant swine, the lifespan of the CLs must be extended to support embryonic and foetal development and vascularisation is necessary for the maintenance of luteal function. Among the molecules involved in the angiogenesis, Vascular Endothelial Growth Factor (VEGF) is the main regulator, promoting endothelial cells proliferation, differentiation and survival as well as vascular permeability and vessel lumen formation. During vascular invasion and apoptosis process, the remodelling of the extracellular matrix is essential for the correct evolution of the CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). Another important factor that plays a role in the processes of angiogenesis and angioregression during the CL formation and luteolysis is the isopeptide Endothelin-1 (ET-1), which is well-known to be a potent vasoconstrictor and mitogen for endothelial cells. The goal of the present thesis was to study the role and regulation of vascularisation in an adult vascular bed. For this purpose, using a precisely controlled in vivo model of swine CL development and regression, we determined the levels of expression of the members of VEGF system (VEGF total and specific isoforms; VEGF receptor-1, VEGFR-1; VEGF receptor-2, VEGFR-2) and ET- 1 system (ET-1; endothelin converting enzyme-1, ECE-1; endothelin receptor type A, ET-A) as well as the activity of the Ca++/Mg++-dependent endonucleases and gelatinases (MMP-2 and MMP-9). Three experiments were conducted to reach such objectives in CLs isolated from ovaries of cyclic, pregnant or fasted gilts. In the Experiment I, we evaluated the influence of acute fasting on VEGF production and VEGF, VEGFR-2, ET-1, ECE-1 and ET-A mRNA expressions in CLs collected on day 6 after ovulation (midluteal phase). The results indicated a down-regulation of VEGF, VEGFR-2, ET-1 and ECE-1 mRNA expression, although no change was observed for VEGF protein. Furthermore, we observed that fasting stimulated steroidogenesis by luteal cells. On the basis of the main effects of VEGF (stimulation of vessel growth and endothelial permeability) and ET-1 (stimulation of endothelial cell proliferation and vasoconstriction, as well as VEGF stimulation), we concluded that feed restriction possibly inhibited luteal vessel development. This could be, at least in part, compensated by a decrease of vasal tone due to a diminution of ET-1, thus ensuring an adequate blood flow and the production of steroids by the luteal cells. In the Experiment II, we investigated the relationship between VEGF, gelatinases and Ca++/Mg++-dependent endonucleases activities with the functional CL stage throughout the oestrous cycle and at pregnancy. The results demonstrated differential patterns of expression of those molecules in correspondence to the different phases of the oestrous cycle. Immediately after ovulation, VEGF mRNA/protein levels and MMP-9 activity are maximal. On days 5–14 after ovulation, VEGF expression and MMP-2 and -9 activities are at basal levels, while Ca++/Mg++-dependent endonuclease levels increased significantly in relation to day 1. Only at luteolysis (day 17), Ca++/Mg++-dependent endonuclease and MMP-2 spontaneous activity increased significantly. At pregnancy, high levels of MMP-9 and VEGF were observed. These results suggested that during the very early luteal phase, high MMPs activities coupled with high VEGF levels drive the tissue to an angiogenic phenotype, allowing CL growth under LH (Luteinising Hormone) stimulus, while during the late luteal phase, low VEGF and elevate MMPs levels may play a role in the apoptotic tissue and extracellular matrix remodelling during structural luteolysis. In the Experiment III, we described the expression patterns of all distinct VEGF isoforms throughout the oestrous cycle. Furthermore, the mRNA expression and protein levels of both VEGF receptors were also evaluated. Four novel VEGF isoforms (VEGF144, VEGF147, VEGF182, and VEGF164b) were found for the first time in swine and the seven identified isoforms presented four different patterns of expression. All isoforms showed their highest mRNA levels in newly formed CLs (day 1), followed by a decrease during mid-late luteal phase (days 10–17), except for VEGF182, VEGF188 and VEGF144 that showed a differential regulation during late luteal phase (day 14) or at luteolysis (day 17). VEGF protein levels paralleled the most expressed and secreted VEGF120 and VEGF164 isoforms. The VEGF receptors mRNAs showed a different pattern of expression in relation to their ligands, increasing between day 1 and 3 and gradually decreasing during the mid-late luteal phase. The differential regulation of some VEGF isoforms principally during the late luteal phase and luteolysis suggested a specific role of VEGF during tissue remodelling process that occurs either for CL maintenance in case of pregnancy or for noncapillary vessel development essential for tissue removal during structural luteolysis. In summary, our findings allow us to determine relationships among factors involved in the angiogenesis and angioregression mechanisms that take place during the formation and regression of the CL. Thus, CL provides a very interesting model for studying such factors in different fields of the basic research.
Resumo:
Eine funktionell und strukturell diverse Gruppe von Transmembranproteinen wie beispielsweise Mediatoren und deren Rezeptoren können proteolytisch gespalten werden. Dieser Prozess wird als Shedding bezeichnet. Kürzlich konnte die proteolytische Aktivität identifiziert werden, die für die Prozessierung von proTNFa verantwortlich ist. Sie wurde TACE (TNF Alpha Converting Enzyme) genannt. In Experimenten mit TACE-/- Fibroblasten konnte ich herausfinden, dass das durch PMA induzierte Shedding des IL-6Rs stark reduziert war. Eine basale hydroxamatsensitive Freisetzung des IL-6Rs konnte allerdings noch detektiert werden. Um Unterschiede im Shedding von IL-6R und proTNFa zu untersuchen, generierte ich chimäre Proteine aus diesen beiden Proteinen, bei denen die Spaltstellenregionen gegeneinander vertauscht worden waren. TNFa Chimären zeigten nur sehr geringes Shedding. Im Gegensatz dazu wurden IL-6R Chimären, die die proTNFa Spaltstelle enthielten spontan gespalten. Die PMA-Induzierbarkeit war verloren gegangen. Daraufhin wurden verschiedene Chimären des unspaltbaren Proteins gp130 und der Spaltstellenpeptide aus TNFa, TGFa und IL-6R generiert. Hierbei wurde ein kurzes membranproximales Peptid aus gp130 gegen die Spaltstellen ausgetauscht. Diese Peptide übertrugen sowohl spontane ale auch PMA-induzierte Spaltbarkeit auf gp130. Um die minimalen Bedingungen für Shedding zu untersuchen, setzte ich verkürzte IL-6R Spaltstellenpeptide in gp130 ein. Die resultierenden Chimären waren empfänglich für reguliertes Shedding. Überaschenderweise konnten auch spaltbare Chimären durch das Ersetzen der membrannahem Region von gp130 durch die entsprechende Region aus dem ebenfalls nicht spaltbaren LIFR generiert werden.
Resumo:
Zusammenfassung In der vorliegenden Arbeit wurden im Zuge derAjmalinbiosynthese in Rauvolfia serpentina die NADPH2abhängigen Reduktionsschritte des Alkaloids Vomilenin zu 17O Acetylnorajmalin genauer untersucht.Dabei konnte erstmals die exakte Reaktionsreihenfolgeaufgedeckt und die daran beteiligten Enzyme ausPflanzenzellsuspensionskulturen isoliert und aufgereinigtwerden. Die ausgearbeiteten, optimierten Reinigungsprotokolleführten in wenigen Stufen gezielt zu den voneinandergetrennten Reduktase Fraktionen. Durch die Trennung derReduktase Aktivitäten war der Grundstein gelegt, dasZwischenprodukt der Reaktion anzureichern und mitverschiedenen analytischen Verfahren als 2?-(R)-1.2Dihydrovomilenin zu identifizieren. Die daraufhin vergebenenBezeichnungen Vomilenin Reduktase (EC.1.5.1.32) und 1.2Dihydrovomilenin Reduktase (EC.1.3.1.73) zielen auf dasumzusetzende Substrat ab.Für die Vomilenin Reduktase konnte eine 4 stufige Reinigungüber (NH4)2SO4 Fällung, Anionen-austauschchromatographie mitSOURCE 30Q, Hydrophobe Interaktionschromatographie an SOURCE15Phe und Affinitätschromatographie mit 2,5 ADP Sepharoseausgearbeitet werden. Hierbei konnte die 1.2Dihydrovomilenin Reduktase schon nach dem erstensäulenchromatogra-phischen Schritt (S 30Q) abgetrenntwerden. Das am Ende der Proteinreinigung angefertigte SDSGel zeigte nur noch 3 Banden, von denen die beiden bei ca.40 und 43 kDa gelegenen Banden mit dem Aktivitätsverlaufder Vomilenin Reduktase korrelierten. Diese wurden einempartiellen Verdau mit der Endoproteinase LysC unterworfen,wobei jeweils 2 Spaltpeptide erhalten werden konnten.Die 1.2 Dihydrovomilenin Reduktase Reinigung umfaßte 6Reinigungsschritte mit (NH4)2SO4-Fällung, SOURCE 30QAnionenaustauschchromatographie, HydroxylapatitChromatographie, 2,5 ADP Sepharose-Chromatographie,Anionenaustausch an DEAE Sepharose und abschließen-denAnionenaustausch über MonoQ. Die resultierendeProteinfraktion wies eine ca. 200 fache Anreicherung an 1.2Dihydrovomilenin Reduktase auf. Auch hierbei wurde die nachSDS Gelelek-trophorese als 1.2 Dihydrovomilenin Reduktasebestimmte Proteinbande (bei ca. 48 kDa) sequen-ziert. Eskonnten vier Peptidfragmente erhalten werden, die ebenso wiedie sequenzierten Peptidstücke der 40 und 43 kDa Bande einehohe Homologie zu Oxidoreduktasen, im einzelnen zuCinnamoylalcohol- und Mannitol Dehydrogenasen, aufwiesen.Um die Identität der sequenzierten Proteinbanden zubestätigen, wurde über reverse genetics die jeweilscodierende cDNA eruiert. Dafür wurden - ausgehend von denPeptidstücken der Mikro-sequenzierung - degenerierte Primerentwickelt und über PCR Teilbereiche der cDNA amplifiziert.Diese konnten für eine radioaktive Durchmusterung einerRauvolfia cDNA Bank herangezogen werden. Alternativ botallein die Kenntnis der spezifischen Nukleotidabfolge dieMöglichkeit der Gewinnung von 5 und 3 Ende derVollängenklone durch RACE PCR.Nach Abschluß dieser Arbeiten konnten für die 40 und 48 kDaBande je ein Vollängenklon und für die 43 kDa Bande 2Vollängenklone (Isoformen) gefunden werden. SämtlicheVollängenklone besitzen einen offenen Leserahmen, der durchnicht zu translatierende Bereiche am 5 und 3 Endeeingefaßt wird. Um die entsprechenden Proteine produzierenzu können, mußten die dafür codierenden cDNA Bereiche dereinzelnen Klone in ein geeignetes Vektor Wirt-System(Expressionssystem) eingebracht werden.Nach erfolgreicher Umklonierung wurde die Expression durchIPTG Zugabe kontrolliert und Proteinrohextrakte aus denBakterienstämmen isoliert. Als Substrate wurden Vomilenin,das strukturisomere Alkaloid Perakin und aufgrund derHomologien zu Cinnamoylalcohol und Mannitol Dehydrogenasen Zimtaldehyd, Dihydrozimtaldehyd und D(-)Fructose getestet. In allen E. coli Stämmen konnte ein unspezifischesReduktionspotential nachgewiesen werden, ohne daß jedochVomilenin reduziert wurde. Die Testung der 1.2Dihydrovomilenin Reduktase Klone mußte wegen Substratmangelentfallen.Die weitere Charakterisierung der pflanzlichen Enzymeerbrachte eine enorm hohe Substratspezifität mit einer sichauf Rauvolfia beschränkenden taxonomischen Verbreitung.Die Molekulargewichtsbestimmung für die Vomilenin Reduktaseergab nach Größenausschluß-chromatographie an Superdex 75ein Gewicht von etwa 43 kDa. Das ebenfalls über Superdex 75ermittelte Molekulargewicht für die 1.2 DihydrovomileninReduktase lag bei ca. 49.8 kDa. Weiterhin wurde eine Metallionenabhängigkeit für dieVomilenin Reduktase aufgezeigt und die Cofaktorspezifitätsowie die pH und Temperatur Optima für beide Reduktasenbestimmt.
Resumo:
In a large number of problems the high dimensionality of the search space, the vast number of variables and the economical constrains limit the ability of classical techniques to reach the optimum of a function, known or unknown. In this thesis we investigate the possibility to combine approaches from advanced statistics and optimization algorithms in such a way to better explore the combinatorial search space and to increase the performance of the approaches. To this purpose we propose two methods: (i) Model Based Ant Colony Design and (ii) Naïve Bayes Ant Colony Optimization. We test the performance of the two proposed solutions on a simulation study and we apply the novel techniques on an appplication in the field of Enzyme Engineering and Design.
Resumo:
Kristallisation der Arbutin-Synthase und der Strictosidin Glukosidase - zwei Enzyme aus dem sekundären Glykosidstoffwechsel von Rauvolfia serpentina Die vorliegende Arbeit befasst sich mit der Kristallisation und der strukturellen Auswertung der Arbutin-Synthase (AS) und der Strictosidin Glukosidase (SG). Beide Enzyme stammen aus der Medizinalpflanze Rauvolfia serpentina. Für die Kristallisation der Arbutin-Synthase wurden ca. 2500 verschiedene Beding-ungen experimentell untersucht. Für einige dieser Experimente wurde das Enzym molekularbiologisch und chemisch verändert. Trotzdem konnten keine Kristalle erhalten werden. Die bei diesen Veränderungen erhaltenen Ergebnisse wurden anhand von Vergleichen mit Strukturen anderer Glykosyltransferasen der gleichen Familie analysiert. Bei der Reinigung der AS konnte mit verschiedenen Trennsystemen nie eine homogene Lösung produziert werden. Der wahrscheinliche Grund für diese schlechte Isolierbarkeit, und damit der wahrscheinliche Grund für die schwierige Kris-tallisation, liegt in der überdurchschnittlich hohen Anzahl an Cysteinen in der Proteinsequenz. Mit den Aminosäuren Cys171, Cys253 und Cys461 wurden drei Cysteine gefunden, die einem Strukturvergleich nach an der Proteinoberfläche liegen und möglicherweise durch Quervernetzungen mit anderen Proteinmolekülen ein heterogenes Gemisch bilden, das nicht geordnet kristallisieren kann. Durch gezielte Mutationen dieser drei Aminosäuren könnte die Kristallisation zukünftig ermöglicht werden. Für die SG waren bereits Bedingungen bekannt bei denen nicht vermessbare Enzymkristalle (Nadeln) wuchsen. In weit gefächerten Versuchen konnten diese Kristalle jedoch nicht zu 3D-Wachstum angeregt werden. Es wurden mit einem HTS-Screening neue Bedingungen zur Kristallisation gefunden. Anschließend konnten die native Struktur und der Strictosidin/Enzym-Komplex vermessen und aufgeklärt werden. Die SG gehört zur Familie 1 der Glukosidasen (GH-1) und besitzt die in dieser Familie konservierte (beta/alpha)8-Barrel-Faltung. Im Vergleich mit 16 bekannten Glykosidasen der Familie GH-1 wurde die Substratbindung untersucht. Dabei wurde die in der Familie konservierte Zuckerbindung vorgefunden, jedoch große Unterschiede in der Aglykonbindung entdeckt. Es wurden Bedingungen für die Konformationsänderung des Trp388 erkannt. Diese Konformationsänderung dirigiert den Aglykonteil des Substrates auf verschiedene Seiten der Substratbindungstasche und teilt so die Familie GH-1 in zwei Gruppen.
Resumo:
In der vorliegenden Arbeit werden verschiedene Enzyme des Ajmalin-Biosynthesewegs aus der Arzneipflanze Rauvolfia serpentina charakterisiert. Dabei handelt es sich einerseits um die Vomilenin-Reduktase und die 2β-(R)-1.2-Dihydrovomilenin-Reduktase. Es wurden Versuche unternommen, diese Enzyme heterolog zu exprimieren. Eine aktive Expression konnte nicht durchgeführt werden, was mit großer Wahrscheinlichkeit auf Modifikationen in der Ursprungspflanze zurückzuführen ist. Allerdings bestehen auch Zweifel, ob es sich bei den Volllängenklonen um die cDNAs der Reduktasen handelte. Zum anderen sollte eine Strukturaufklärung der Vinorin-Synthase im Komplex mit Liganden vorgenommen werden. Die erhaltenen Proteinkristalle stellten sich als derart empfindlich gegenüber Schwankungen ihrer Umgebung und dem Eindringen von Liganden in den Kristall dar, dass eine erfolgreiche Komplexierung und strukturelle Beschreibung durch Röntgenstrukturanalyse nicht möglich war. Weiterhin wurden Mutagenesestudien mit der Vinorin-Synthase durchgeführt. Eine Asparaginsäure bildet eine Salzbrücke mit einem Arginin. Alle durchgeführten Mutationen dieser Asparaginsäure führten zu einem absoluten Aktivitätsverlust. Eine Funktion des Asparagins 277, als mitverantwortliche Aminosäure zur Bindung des Co-Substrats Acetyl-CoA, konnte anhand der Mutagenesestudien ausgeschlossen werden. Weiterhin ist es erstmals gelungen die Polyneuridinaldehyd-Esterase aus Rauvolfia serpentina zu kristallisieren. Schließlich konnte die dreidimensionale Struktur der Polyneuridinaldehyd-Esterase aufgeklärt werden. Es folgte eine Beschreibung struktureller Eigenschaften der Polyneuridinaldehyd-Esterase im Vergleich zu einem Modell, welches durch ein „Molecular Modelling“ erstellt wurde.