Η σεργλυκίνη (SG) είναι η κύρια πρωτεογλυκάνη που εκφράζεται από τα αιμοποιητικά κύτταρα και συμμετέχει στη ρύθμιση διαφόρων παραγόντων που εμπλέκονται στις αντιδράσεις φλεγμονής. Επιπλέον, φαίνεται ότι διαδραματίζει σημαντικό ρόλο στη βιολογία του πολλαπλού μυελώματος αφού αναστέλλει τη δραστικότητα της κλασικής οδού και της λεκτινικής οδού του συμπληρώματος και προάγει την προσκόλληση των μυελωματικών κυττάρων στο κολλαγόνο τύπου Ι. Παράλληλα, η αυξημένη έκφρασή της σχετίζεται με τον επιθετικό φαινότυπο των κυττάρων καρκίνου του ρινοφάρυγγα. Στην παρούσα διατριβή μελετήθηκε η έκφραση της SG σε κακοήθειες. Τα αποτελέσματά μας αναδεικνύουν την έντονη παρουσία της σε συμπαγείς όγκους λόγω της αυξημένης έκφρασή της είτε από τα καρκινικά κύτταρα είτε από τα κύτταρα φλεγμονής και τα κύτταρα του στρώματος τα οποία επιστρατεύονται κατά την ανάπτυξη του όγκου. Επιπλέον, η αυξημένη έκφρασή της και η έκκρισή της σχετίσθηκαν με το μεταστατικό δυναμικό των κυτταρικών σειρών διαφόρων τύπων καρκίνου. Παράλληλα, ταυτοποιήθηκε η έκφραση του μεταγράφου της SG που προκύπτει από εναλλακτικό μάτισμα με απαλοιφή του εξωνίου 2. Επιπροσθέτως, μελετήθηκε ο βιολογικός ρόλος της SG σε επιθετικά καρκινικά κύτταρα μαστού. Βρέθηκε ότι η SG αλληλεπιδρά με πρωτεΐνες που διαδραματίζουν σημαντικό ρόλο σε κυτταρικές λειτουργίες όπως η αναδιοργάνωση του κυτταροσκελετού, η μεταφορά και ανακύκλωση μορίων από και προς την κυτταρική επιφάνεια και η γονιδιακή ρύθμιση. Η ίδια πρωτεογλυκάνη εκκρίνεται ιδιοσυστατικά από αυτά τα κύτταρα. Τόσο η γλυκοζαμινογλυκανική της σύσταση όσο και η ανασταλτική της δράση έναντι της ενεργοποίησης του συστήματος του συμπληρώματος είναι παρόμοιες με τη SG που εκκρίνεται από τα μυελωματικά κύτταρα. Η συμβολή της SG στον επιθετικό φαινότυπο των καρκινικών κυττάρων επιβεβαιώθηκε με την υπερέκφρασή της σε χαμηλής επιθετικότητας κύτταρα καρκίνου του μαστού. Λειτουργίες όπως ο κυτταρικός πολλαπλασιασμός, η μετανάστευση και η διήθηση των καρκινικών κυττάρων σχετίσθηκαν θετικά με την αυξημένη έκφραση και έκκριση της SG από αυτά τα κύτταρα. Οι επαγωγικές της ιδιότητες καταργήθηκαν με την απαλοιφή των θέσεων πρόσδεσης των γλυκοζαμινογλυκανών από τον πρωτεϊνικό της κορμό. Επιπλέον, διερευνήθηκε το πρωτεολυτικό δυναμικό αυτών των κυττάρων με τη μελέτη της έκφρασης πρωτεολυτικών ενζύμων του εξωκυττάριου χώρου, όπως οι tPA, uPA, MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP και του αναστολέα των μεταλλοπρωτεϊνασών TIMP-1. Παρουσιάσθηκε ότι η υπερέκφραση του πλήρους μορίου της SG αλλά και της μη γλυκοζυλιωμένης της μορφής οδηγούν στη διαφοροποίηση της έκφρασης και της ενεργότητας των μορίων αυτών με τρόπο που ποικίλει ανάλογα με την κυτταρική πυκνότητα. Παρόλα αυτά, τα δεδομένα που προέκυψαν από τη μελέτη της έκφρασης του uPA και της MT1-MMP συσχέτισαν την αυξημένη τους ενεργότητα με την υπερέκφραση της γλυκοζυλιωμένης μορφής της SG και ανέδειξαν την πιθανή συμβολή τους στην επιθετικότητα των καρκινικών κυττάρων μαστού. Συμπερασματικά, η παρουσία της SG είναι έντονη σε πληθώρα συμπαγών όγκων και καρκινικών κυτταρικών σειρών και φαίνεται να σχετίζεται με το μεταστατικό δυναμικό των κυττάρων. Η συμβολή της στον επιθετικό φαινότυπο των καρκινικών κυττάρων περιλαμβάνει τόσο ενδοκυττάριες όσο και εξωκυττάριες δράσεις που μεσολαβούνται από τη γλυκοζυλιωμένη της μορφή. / Serglycin (SG) is the major proteoglycan of hematopoietic origin cells and contributes to the regulation of several inflammatory proteins. Moreover, SG has a significant role in the biology of Multiple Myeloma; It inhibits the activation of the classical and the lectin pathway of complement system. Enhanced SG expression is correlated with the aggressive phenotype of nasopharyngeal cancer cells. In the present study, the expression of SG in several malignancies was investigated. The strong presence of SG in solid tumors due to its elevated expression either by cancer cells or by inflammatory and stomal cells was revealed. Furthermore, SG elevated expression and secretion was correlated with the high metastatic potential of several cancer cell lines. The expression of the alternatively spliced SG mRNA (variant 2) was identified. This variant lacks exon 2. The role of SG in the biology of aggressive breast cancer cells was studied. SG interacts with significant mediators of actin cytoskeleton reorganization, protein transport and regulation of gene expression. This proteoglycan is constitutively secreted by aggressive breast cancer cells and shares the same glycosaminoglycan (GAG) moieties and inhibitory effects torward complement system activation as the secreted SG by myeloma cells. SG contribution in the aggressive phenotype of cancer cells was studied via the overexpression of the moleclule in the low aggressive breast cancer cells. Cellular functions such as proliferation, migration and invasion of cancer cells were positively correlated with the elevated expression and secretion of SG. These properties were abolished by the deletion of GAG binding sites from SG core protein. Moreover, the proteolytic potential of the overexpressing cells was examined via the expression of ECM degrading molecules, such as tPA, uPA, MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP, and TIMP-1 MMP inhibitor. Altered expression and activity rates of these enzymes correlated with the overexpression of either the full length or the truncated form of SG in a manner which depends on the cellular density. Interestingly, enhanced MT1-MMP activity followed only the overexpression of the full length molecule indicating the contribution of this MMP in breast cancer cell aggressiveness. The data above revealed the intense presence of SG in several solid tumors and cancer cell lines and the correlation of SG expression with the metastatic potential of cancer cells. Its contribution to cancer cells aggressive phenotype includes both intracellular and extracellular functions which are mediated by the glycanated form of SG.

Biofilme sind eine der widerstandsfähigsten Formen mikrobiellen Lebens. Ihr frühzeitiges Auftreten in der Erdgeschichte konnte durch Stromatolithfunde bewiesen werden. Heutige Biofilme und mikrobielle Matten bieten somit eine Möglichkeit wichtige Einblicke und Erkenntnisse über das erste Leben auf unserem Planeten zu geben. In dieser Arbeit wurden die prokaryotischen Lebensgemeinschaften von verschiedenen Ökosystemen mittels metagenomischer und metatranskriptomischer Methoden analysiert. Mithilfe von „Next-Generation Sequencing“ wurden 16S rRNA Genanalysen, metatranskriptomische Analysen und funktionsbasierte Durchmusterungen von Fosmid-Metagenombanken durchgeführt. Die bakterielle Zusammensetzung und Diversität von kalzifizierenden Biofilmen und dem unterliegenden Kalktuff des Frischwasserbachs Westerhöfer Bach wurden analysiert. Es konnte gezeigt werden, dass der Biofilm hauptsächlich von filamentösen Cyanobacteria, aeroben Vertretern aus allen Klassen der Proteobacteria und Chloroflexi bevölkert wurde. Die bakterielle Diversität nahm flussabwärts zu, was auf Änderungen der physikochemischen Parameter zurückgeführt wird. Aufgrund geringerer UV-Einstrahlung waren im Kalktuff mehr Proteobacteria als Cyanobacteria vorhanden. Des Weiteren gab es deutliche Unterschiede zwischen den relativen Abundanzen der gesamten und aktiven proteobakteriellen Klassen im Biofilm. Die aktiven Funktionen der Biofilm-Mikrobiota einer Westerhöfer Bach Probe wurden mittels metatranskriptomischer Methoden genauer analysiert. Die meisten Transkripte der mikrobiellen Biofilmgemeinschaft umfassten Gene der Photosynthese, des Proteinmetabolismus, des Kohlenstoffmetabolismus und der Zellatmung. Um das metagenomische Potential des Westerhöfer Bach Biofilms zu erschließen, wurden vier „large-insert“ Metagenombanken konstruiert. Funktionsbasierende Durchmusterungsverfahren führten zur Identifikation von fünf bisher unbekannten Genen, die für proteolytische Enzyme kodieren und einem Gen-Cluster, welches für cellulolytische Enzyme kodiert. Bei dem zweiten untersuchten Habitat handelt es sich um eine mikrobielle Matte des hypersalinen Lake 21 auf Kiritimati. Die Mikrobialith-bildende Matte besteht aus neun klar abgegrenzten, unterschiedlich gefärbten Lagen, welche separat auf ihre bakterielle und archaelle Zusammensetzung analysiert wurden. Anhand der prokaryotischen Zusammensetzung und dem Sauerstoff- und Lichtgradienten ergab sich eine Einteilung der mikrobiellen Matte in drei Zonen. Im Allgemeinen erhöhte sich die prokaryotische Diversität mit Tiefe der Matte, wohingegen das Redoxpotential und der pH-Wert sanken. Passend zu den hydrochemischen Daten änderte sich die prokaryotische Zusammensetzung von der photisch-oxischen Zone, welche aus halophilen, oxygenen und anoxygenen Phototrophen und aeroben Heterotrophen bestand, zu Sulfat-reduzierenden Bakterien (SRB), Fermentierern und potentiell Sulfat-reduzierenden Archaeen in der Übergangszone. In der anoxischen Zone konnten hauptsächlich SRB, Fermentierer, Ammonium-oxidierende Archaea und geringe Mengen methanogene Archaeen detektiert werden. Von den kenianischen Natronseen Bogoria, Sonachi, Elementeita und Magadi wurde die prokaryotische Zusammensetzung und Diversität von Boden-, Sediment-, Wasser-, und mikrobiellen Mattenproben analysiert. Hier zeigte sich, dass Boden- sowie Sedimentproben hauptsächlich von Proteobacteria, Gemmatimonadetes, Firmicutes, Actinobacteria, Acidobacteria und Bacteroidetes bevölkert wurden, wohingegen in den Wasserproben Cyanobacteria vorherrschten. Die Archaeen wurden überwiegend von unterschiedlichen Vertretern der Halobacteria repräsentiert. In den humiden Proben wurden außerdem methanogene Archaeen und Thaumarchaeota nachgewiesen. Letztlich wurde in dieser Arbeit die bakterielle Zusammensetzung des Biofilms und des dazugehörigen Planktons von mikrobiellen Brennstoffzellen (MBZ) untersucht. Der erzeugte Datensatz demonstrierte, dass die aktive und gesamte bakterielle Lebensgemeinschaft in den einzelnen Replikaten minimal variierte. Generell zeigte sich, dass stromproduzierende MBZ eine niedrigere bakterielle Diversität aufwiesen als nicht stromproduzierende MBZ. Des Weiteren zeigte die Analyse, dass bisher unkultivierte Vertreter der Spezies Geobacter und Clostridium mit der Stromproduktion verbunden waren.

Dyslipidemia can be a comorbidity of both insulin-resistance and atherosclerosis. Hypertriglyceridemia is common in hyperglycemia and is associated with hypoalphalipoproteinemia (low HDL) and with altered nucleotide or purinergic signaling. We therefore hypothesized that extracellular nucleotides may affect hepatic lipoprotein metabolism. Our studies confirm this view and show that nucleotides regulate cellular proteolytic pathways in liver cells and thereby control lipoprotein secretion and their metabolism by hepatic lipase (HL). Treatment of liver cells with the nucleotide, adenosine diphosphate (ADP), stimulates VLDL-apoB100 and apoE secretion, but blocks HDL-apoA-I and HL secretion. ADP functions like a proteasomal inhibitor to block proteasomal degradation and stimulate apoB100 secretion. Blocking the proteosome is known to activate autophagic pathways. The nucleotide consequently stimulates autophagic degradation in liver cells and increases cellular levels of the autophagic proteins, LC3 and p62. Confocal studies show that ADP increases cellular LC3 levels and promotes co-localization of LC3 and apoA-I in an autophagosomal degradation compartment. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy and block both HDL and HL secretion. Overexpression of P2Y13 increases cellular LC3 levels and blocks the induction of both HDL and HL secretion, while P2Y13 siRNA reduce LC3 protein levels and cause up to a ten-fold stimulation in HDL and HL secretion. P2Y13 gene expression regulates autophagy through the insulin receptor (IR-β). A reduction in P2Y13 expression increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, while increasing P2Y13 expression inhibits the activation of IR-β and Akt. Experiments with epitope-labeled apoA-I and HL show that activation of purinergic pathways has no effect on the internalization and degradation of extracellular apoA-I and HL, which confirms the view that nucleotides primarily impact intracellular protein transport and degradation. In conclusion, elevated blood glucose levels may promote dyslipidemia by stimulating purinergic signaling through P2Y13 and IR-β and perturbing the intracellular degradation and secretion of both HDL and VLDL.

Die Dicklegung der Milch, ausgehend von der hydrolytischen Spaltung des κ-Caseins, stellt den ersten essentiellen Schritt in der Käseherstellung dar. Dabei finden Gerinnungsenzyme verschiedener Herkunft Anwendung, deren neueste Varianten auf Grundlage des aktuellen Forschungsstandes umfassend charakterisiert werden. Verschiedene Kälberlabpräparate, mikrobielle Gerinnungsenzyme aus Rhizomucor miehei und mit Hilfe von gentechnisch modifizierten Mikroorganismen gewonnenes Chymosin (FPC) aus Rind und Kamel werden mittels HPLC und Elektrophorese hinsichtlich ihrer Zusammensetzung analysiert. Die neueste Generation mikrobieller Enzyme weist im Gegensatz zur herkömmlichen Variante keine Nebenkomponenten und damit einen höheren Aufreinigungsgrad auf. Die unspezifische proteolytische Aktivität wird durch fluorimetrische Quantifizierung der in 12 % TCA löslichen Stickstoffkomponenten bestimmt, die nach Inkubation rekonstituierter Magermilch bei 32 °C und pH 6,5 über 24 h mit den Enzymen freigesetzt werden. Mikrobielle Gerinnungsenzyme besitzen eine signifikant höhere unspezifische proteolytische Aktivität gegenüber chymosinbasierten Präparaten, deren Auswirkung sich bei Erhöhung der zugegebenen Enzymmenge besonders ausgeprägt darstellen. Oszillationsrheometrische Analysen lassen bei gleicher Enzymaktivität eine geringere Gelfestigkeit nach 40 min bei Einsatz von mikrobiellen Präparaten im Vergleich zu Kälberlab und bovinem FPC erkennen. Zusätzlich wird eine Abhängigkeit der Flockungszeit und der Gelfestigkeit vom eingesetzten Substrat beobachtet, die für Chymosin aus Kamel besonders stark ausgeprägt ist. Die Substratspezifität dieses Enzyms ist weder mit der des bovinen Chymosins noch mit der der mikrobiellen Gerinnungsenzyme identisch. Im Rahmen von Käsungsversuchen im Labor-, Pilot- und Industriemaßstab wird eine signifikant höhere Käseausbeute (0,50 - 1,19 %) bei Verwendung vom traditionellem Kälberlab im Vergleich zur neuesten Generation der kommerziellen mikrobiellen Substitute ermittelt. Im Verlaufe der Reifung von Schnittkäse wird durch mikrobielles Gerinnungsenzym gegenüber Kälberlab eine signifikant höhere Menge an Nichtproteinstickstoff freigesetzt sowie ein unterschiedliches Profil an Proteinabbauprodukten gebildet. Die höhere proteolytische Aktivität resultiert in einer signifikant stärker ausgeprägten Bitterkeit der mit mikrobiellem Gerinnungsenzym hergestellten Käse nach 12 Wochen Reifungszeit. / Clotting of milk caused by hydrolytic cleavage of κ-casein is the first important step in cheese milk processing. This cleavage is caused by clotting enzymes of different origin, which are comprehensively characterized by considering results of latest investigations. The composition of selected calf rennets, microbial coagulants derived from Rhizomucor miehei and genetically engineered chymosin (FPC) derived from cow and camel is analyzed by HPLC and electrophoresis. In contrast to conventional products, the latest generation of microbial coagulants does not show minor components in a detectable amount because of a sufficient purification. The unspecific proteolytic activity is determined by fluorimetric quantification of 12 % tricloric-acid-soluble nitrogen, which is released by the enzymes from reconstituted skim milk, pH 6.5, after incubation at 32 °C for 24 h. Microbial coagulants show a significantly higher unspecific proteolysis as compared to chymosin-based clotting enzymes, especially when the enzymes are added in amount higher than used during cheese-making. Small amplitude oscillation rheometry analysis showed a lower gel firmness after 40 min of gelling when microbial coagulants were applied instead of calf rennet or FPC. Furthermore, flocculation time, gel formation time and gel firmness additionally depends on the test substrate, and this dependency is exceptionally pronounced when camel chymosin was used. The substrate specificity of this enzyme is neither identical to that of bovine chymosin nor to that of microbial coagulants. Cheese making experiments in laboratory-, pilot- and commercial-scale revealed a significantly higher cheese yield (0.50 - 1.19 %) when using calf rennet instead of microbial coagulant of the latest generation. During ripening of semi-hard cheese a higher amount of non-protein-nitrogen was released and a different electrophoretic casein degradation profile was generated when using microbial enzymes. Enhanced proteolysis is responsible for a significantly higher pronounced bitterness of microbial produced cheese after 12 weeks of maturation.

Die Bildung von Amyloidablagerungen ist ein Kennzeichen der Alzheimerschen Erkrankung. Hauptbestandteil dieser senilen Plaques sind sogenannte A beta Peptide, die durch proteolytische Prozessierung aus einem Vorläufermolekül (APP) gebildet werden. Die vorliegende Arbeit beschreibt die Klonierung des Meerschweinchen - APP. Diese cDNA-Sequenz zeigt auf DNA-Ebene eine Homologie zum Human-APP von ca. 90%, auf Proteinebene beträgt die Identität ca. 97 %. Damit wird ein weiterer experimenteller Beweis für die evolutionäre Konservierung des Amyloidvorläuferproteins in Säugetieren erbracht. APP mRNA wird in Meerschweinchen-Geweben ubiquitär exprimiert. Durch alternatives Spleißen wird ein zum Human-APP im wesentlichen ähnliches Isoformenmuster gebildet: Isoformen, welche eine Proteaseinhibitordomäne enthalten, werden dominierend in peripheren Organen exprimiert, dagegen ist im Zentralnervensystem das APP 695 mit über 60 % der Gesamttranskripte die bevorzugt exprimierte Isoform. Die klonierte cDNA des Meerschweinchen-APP wurde in prokaryontischen wie auch eukaryontischen Zellsystemen exprimiert. Dabei wurde die Eignung einer Anzahl von gegen Human-APP gewonnenen Antikörpern zur Detektion des Meerschweinchen-APP und seiner Prozessierungsprodukte gezeigt. Die Expression der neuronal dominierend exprimierten Isoform APP 695 des Meerschweinchen-APP in humanen Neuroblastom-Zellen zeigte keine Unterschiede hinsichtlich der APP-Prozessierung und A beta-Bildung im direkten Vergleich zu Human-APP 695. Die proteolytische Prozessierung des Proteins wurde durch Detektion der typischen Spaltprodukte in vivo (im Liquor) als auch in einem neu etablierten in vitro-Modell primär kultivierter neuronaler Zellen untersucht. Diese Zellkulturen wurden zunächst immunhistochemisch und biochemisch charakterisiert und als "mixed brain"-Typ mit einem hohen neuronalen Anteil beschrieben. Die Prozessierung des endogenen Meerschweinchen-APP in kultivierten Zellen führt dabei zur Bildung und Akkumulation aggregationsfähiger A beta - Peptide. Zur Detektion dieser Peptide wurde ein sensitiver Nachweis durch Western-Blot etabliert. Es wird damit ein Modellsystem für in vitro-Untersuchungen vorgeschlagen, welches ein Studium der Expression und Prozessierung des Amyloidvorläuferproteins unter angenähert physiologischen Bedingungen ermöglicht. / A beta peptides, the major component of neuritic plaques found in the brains of patients with Alzheimer’s disease, are derived by proteolytic processing from a larger precursor molecule (amyloid precursor protein - APP). A combination of PCR methods was used to clone and sequence APP cDNA from guinea pig (Cavia porcellus). Guinea pig APP exhibits extensive similarities to human APP in terms of primary structure, mRNA expression of differentially spliced isoforms as shown by Northern blot and RT-PCR analysis as well as proteolytic processing to amyloidogenic A beta peptides. In contrast to rat and mouse APP, guinea pig APP - recombinantly expressed in human neuroblastoma-cells - was processed indistinguishable from human APP thus excluding intrinsic sequence-specific factors influencing processing. Further studies were performed using newly established primary cell cultures of guinea pig neurons. Refined methods have been used to detect and characterize major proteolytic processing products of APP in vitro and in vivo. In conclusion, guinea pigs provide a model to study expression and processing of APP that closely resembles the physiological situation in humans and should, therefore, be important in elucidating potential strategies to prevent amyloid formation in Alzheimers Disease.

A produção de proteases e biossurfactantes por Bacillus licheniformis UCP-1014 foi investigada neste trabalho. Os experimentos foram realizados em frascos de Erlenmeyer de 125 mL, em triplicata, inóculo 10% v/v, a 150 rpm e 37C. Um planejamento fatorial foi realizado para investigar as concentrações dos componentes do meio de cultivo. Amostras de líquido metabólico foram coletadas, centrifugadas e os sobrenadantes utilizados para determinar pH, atividade proteolítica e tensão superficial. O líquido metabólico foi concentrado por ultrafiltração e a estabilidade da atividade proteolítica no retentado foi determinada quanto ao pH e à temperatura. A estabilidade do retentado foi investigada por planejamento fatorial e a atividade proteolítica determinada com 10, 20 e 30 dias de armazenamento a 28 C. A determinação de proteases foi realizada na presença de azo-caseína. A cultura de B. licheniformis UCP-1014 produziu 112 U/mL de proteases na presença de melaço 1% e uréia 0,5%, a pH 7,5 com 24 h de cultivo. A redução da tensão superficial do líquido metabólico não foi significativa nessas condições de trabalho. O líquido metabólico concentrado reteve cerca de 50% da atividade proteolítica inicial. O concentrado de proteases apresentou a maior atividade enzimática em pH 8 durante 30 min de incubação, retendo 97 % da atividade; a estabilidade térmica máxima foi a 50C durante 30 min, retendo 98 % da atividade enzimática. O retentado do líquido metabólico após formulado manteve 54 % da atividade com 30 dias de armazenamento a 28C. Proteases produzidas por B. licheniformis UCP-1014 na presença de nutrientes de baixo custo podem ser competitivas no mercado / The production of protease and biosurfactant by Bacillus licheniformis UCP-1014 was investigated in this work. The experiments were performed in Erlenmeyer flasks, in triplicate, and inoculum 10% v/v, 150 rpm and 37C. A factorial design was conducted to investigate the concentrations of the medium. Metabolic fluid samples were collected, centrifuged and the supernatant used to determine pH, proteolytic activity and surface tension. The liquid was concentrated by ultrafiltration metabolic the stability and proteolytic activity in the retentate was determined for pH and temperature. In making the retentate was used a factorial design, and protease stability was determined during 10, 20 and 30 days at 28C. The determination of protease was performed in the presence of azo-casein. The culture of B.licheniformis UCP-1014 produced 112 U/mL protease in the presence of 1% molasses and urea 0,5%, pH 7,5 at 24h of culture. The reduction in surface tension was not significant in these metabolic conditions. The concentration of proteases produced by B. licheniformis UCP-1014 had the highest stability of enzyme activity in the absence of substrate at pH 7 during 60 min of incubation and maximum thermal stability between 40 90C for 90 min. The liquid concentrate and formulated metabolic retained about 50% of proteolytic activity whose value decreased during storage at 28C. Proteases produced by B. licheniformis UCP-1014 in the presence of nutrients of low cost can be competitive in the market

O reaproveitamento de resíduos agroindustriais como fontes de carbono e de nitrogênio tem sido investigado na área de biotecnologia para produção de enzimas por micro-organismos. Dentre as enzimas microbianas importadas no Brasil, as proteases são aplicadas no processamento tecnológico de detergentes, fármacos, cosméticos, alimentos, dentre outros. O objetivo deste trabalho foi produzir proteases por cultivo submerso utilizando resíduos agroindustriais. A determinação da atividade proteolítica ocorreu na presença de azocaseína a 0,2 %. Cultivos submersos de culturas isoladas de Bacillus sp. foram realizados em frascos de Erlermeyer. A atividade máxima de proteases foi determinada na presença de milhocina. A concentração do líquido metabólico com atividade proteolítica por ultrafiltração reteve cerca de 80 % da atividade inicial. Um planejamento experimental fatorial foi realizado para investigar a estabilidade do líquido metabólico. A maior atividade proteolítica média do líquido metabólico livre de células (196 U/mL) foi determinada na presença de sorbato de sódio a 0,5 %, cloreto de cálcio a 0,5 %, glicerol a 7,5 % e polietilenoglicol-200 a 0,5 %. O extrato enzimático formulado reteve 68 % da atividade proteolítica com 10 dias de armazenamento à temperatura ambiente (28 ˚C). O retentado com atividade proteolítica apresentou pH ótimo 9 e 11 e retenção de 90 - 100 % da atividade durante 90 min em pH ótimo; a temperatura ótima foi 50 ˚C e a estabilidade térmica máxima a 40 ˚C durante 30 min a pH 11. A formulação de líquido metabólico concentrado com atividade proteolítica que apresenta estabilidade térmica em pH alcalino é um bioproduto que pode ser utilizado como aditivo em detergentes. / The reuse of agro-industrial waste as sources of carbon and nitrogen has been investigated in biotechnology for the production of enzymes by microorganisms. Among the microbial enzymes imported into Brazil, the proteases are applied in technological processes in the fields of detergents, pharmaceuticals, cosmetics, food, among others. The aim of this work was to produce proteases by submerged cultivation in the presence of agro-industrial waste. The determination of proteolytic activity was in the presence of 0.2 % azocasein. Submerged cultivation of Bacillus sp. strains isolated were carried out in Erlermeyer flasks. The maximum protease activity was determined in the presence of corn steep liquor. The concentration of the liquid metabolic by ultrafiltration of the metabolic liquid with proteolytic activity retained 80% of the activity. A factorial experimental design was carried out to investigate the stability of the metabolic liquid. The maximum proteolitic activity of the liquid metabolic cell-free (196 U/mL) was determined in the presence of 0.5 % sodium sorbate, 0.5 % calcium chloride, and 7.5 % of glycerol and polyethyleneglycol-200. The enzyme extract formulated retained 68 % of the proteolytic activity after 10 days at storage at room temperature (28 ˚C). The retentate with proteolytic activity showed optimum pH 9 and 11 and retention 90 - 100 % of the activity for 90 min at optimum pH; the optimum temperature was 50 ˚C e the maximum thermal stability was at 40 ˚C for 30 min at pH 11. The formulation of metabolic liquid concentrate with proteolytic activity which has thermal stability at alkaline pH is a bioproduct which can be used as an additive in detergents.

Orientador: Eleni Gomes / Banca: Luiz Juliano Neto / Banca: João Ruggiero Neto / Resumo: Fungos termofílicos têm despertado grande interesse acadêmico e industrial por produzirem uma variedade de enzimas termoestáveis com potenciais aplicações em processos biotecnológicos como biocatálise nas indústrias de couro, farmacêutica, têxtil e alimentícia, e na preparação de produtos de limpeza e cosméticos. Particularmente, as proteases, além de participarem de inúmeros processos fisiológicos vitais como vias metabólicas, hemostasia e sinalização celular, também representam hoje cerca de 60% do mercado mundial de enzimas. Neste trabalho, descrevemos a produção, purificação e caracterização bioquímica de uma serino-protease produzida por um fungo termofílico do gênero Myceliophthora. As taxas de atividade proteolítica foram avaliadas através de fermentação em meio sólido (FES) e submerso (FSM) e observou-se um rendimento na atividade proteolítica 4,5 vezes maior para o meio sólido. A enzima bruta obtida por ambos os procedimentos (FES e FSM) exibiu a mesma temperatura ótima de 50 ºC, porém em relação ao pH ótimo houve um deslocamento de 7 (FSM) para 9 (FES) sugerindo que o perfil enzimático do fungo difere de acordo com suas condições de fermentação. Baseado nesses resultados prosseguiu-se os estudos com o extrato bruto obtido por FES. A imobilização da enzima bruta em esferas de alginato de cálcio resultou no aumento da temperatura ótima e na estabilidade térmica quando comparado com a enzima livre. O extrato bruto obtido por FES foi, então, fracionado por métodos cromatográficos como exclusão molecular e troca-iônica que resultaram na protease pura com peso molecular de 28,2 kDa determinado por espectrometria de massa. A protease pura demonstrou pH ótimo de 9,0 e temperatura ótima de 45 °C que corroboram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Thermophilic fungi have attracted great academic and industrial interest because they produce a variety of thermostable enzymes with potential applications in biotechnological processes such as biocatalysis in the industries of leather, pharmaceutical, textile and food, and the preparation of detergents and cosmetics. In particular, proteases not only participate in many vital physiological processes such as metabolic pathways, cell signaling and homeostasis, but also currently represent about 60% of the world market of enzymes. In this work, we describe the production, purification and biochemical characterization of a serine protease produced by a thermophilic fungus of the genus Myceliophthora. The levels of proteolytic activity were evaluated either by solid fermentation (SSF) and submerged (SmF). The crude enzyme obtained by both procedures (SSF and SmF) exhibited similar optimum temperature of around 50 ºC, but in relation to the optimum pH was shifted of 7 (SmF) to 9 (SSF), suggesting that the enzymatic profile of the fungus differs from according to its fermentation conditions. Based on these results, the studies were followed with crude extract obtained by SSF. The immobilized enzyme on beads of calcium alginate resulted in increased optimum temperature and thermal stability when compared to the free enzyme. The crude extract obtained by SSF was then fractionated by chromatographic methods including molecular exclusion and ion-exchange that resulted in the pure protease with molecular weight of 28.2 kDa as determined by mass spectrometry. The pure protease showed optimum pH of 9.0 and optimum temperature of 45 °C that corroborate to the preliminary characterization of the crude extract. Inhibition tests resulted in complete inhibition by PMSF, a canonical... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-12-17T15:36:36Z (GMT). No. of bitstreams: 1 MarinoEAN_TESE.pdf: 1510121 bytes, checksum: 824b52d7ab71a45fa42ff2c16c22da7c (MD5) Previous issue date: 2011-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Shrimp farming in Brazil is a consolidated activity, having brought economical and social gains to several states with the largest production concentrated in the northeast. This fact is also reflected in higher feed intake, necessitating a more efficient feed management. Currently, management techniques already foresee food loss due to molting. In this sense, studies relating shrimp s digestive physiology, molting physiology and behavioral response of shrimp feed can optimize the feed management. Thus, our study aimed to evaluate the behavioral response of the marine shrimp L. vannamei (Crustacea: Penaeidae) in accordance with the stages of moulting cycle and feeding schedules based on higher or lower activity of proteolytic digestive enzymes; also, to investigate the influence of feeding schedule on hepatosomatic index and non-specific and specific protease activity (trypsin). Experiments were carried out at the Laboratory of Shrimp Behavioral Studies at UFRN in partnership with the Laboratory of Enzimology UFPE. Juveniles of L. vannamei weighting 5.25 g (+ 0.25 g) were kept in aquaria at a density of 33 shrimp m -2. In the first experiment, shrimp were fed in the light phase or in the dark phase for 8 days; in the ninth day, the animals were observed for 15 minutes every hour during the 12 hours of each phase of the photoperiod. We recorded the frequency of inactivity, exploration, food intake, burrowing, swimming and crawling behavior. At the end of the 12th observation session, the shrimp were sacrified and classified by the method of setogenesis in the molt cycle stages A, B, C, D0, D1, D2 or D3. We found that the shrimp in A stage show high levels of inactivity. Moreover, the frequency of food intake was very low. The shrimp in D3 stage also had low food intake and high inactivity associated with elevated frequencies of burrowing. In the second experiment, shrimp were kept in physiological acclimation to experimental conditions for 28 days, distributed in 12 treatments in the light phase and 12 treatments in the dark phase. In the end, the animals were sacrified and dissected to assess non-specific and specific protease activity (trypsin) activity. In general, these parameters did not vary among animals fed in the light phase and those fed in the dark phase. However, significant differences were found in the activity of specific and nonspecific proteases in relation to food treatment. In the light phase, the major proteolytic activities converged to 10 hours after the start of the light phase, while the lowest activities converged to 6 hours after the beginning of this phase. In the dark phase, the highest enzyme activity converged to 12 hours after the onset of phase, while the lowest activities converged to 3 hours after the onset of phase. In the third experiment, we sought to evaluate the behavioral responses of shrimp in relation to dietary treatments based on higher or lower activity of proteolytic enzymes, considering the results of the second experiment. The behavioral categories observed were the same as the ones in the first experiment, with observations of 30 minutes (15min before and 15min after food supply). We found variation in behavioral responses as a function of the treatments, with greater intake of food in shrimp fed during the period of greatest activity of proteolytic enzymes, in the light phase. Thus we see that periodic events associated with the shrimp s physiology interfere in their behavioral responses, revealing situations that are more adjustable to the provision of food, and consequently optimizing feeding management / A carcinicultura brasileira ? uma atividade consolidada, pois trouxe ganhos econ?micos e sociais a v?rios estados, com a maior produ??o e ?rea cultivada localizada na regi?o nordeste. Esse fato tamb?m se reflete no maior consumo de ra??o, tornando necess?rio um manejo alimentar mais eficiente. Atualmente, as t?cnicas de manejo j? prev?em sobra alimentar em decorr?ncia dos processos de muda. Nesse sentido, estudos relacionando fisiologia digestiva, fisiologia da muda e resposta comportamental do camar?o podem otimizar o manejo alimentar. Assim, nosso trabalho teve o objetivo de avaliar a resposta comportamental do camar?o marinho L. vannamei (Crustacea: Penaeidae) de acordo com os est?gios do ciclo de muda e com a alimenta??o baseada em hor?rios de maior ou de menor atividade de enzimas digestivas proteol?ticas, bem como investigar a influ?ncia do hor?rio de alimenta??o sobre o ?ndice hepatossom?tico, atividade de protease espec?fica (tripsina) e n?o espec?fica. Para tanto, foram realizados tr?s experimentos no Laborat?rio de Estudos do Comportamento de Camar?es Departamento de Fisiologia da UFRN, em parceria com o Laborat?rio de Enzimologia Departamento de Bioqu?mica da UFPE. Juvenis de L. vannamei pesando 5,25 g (+ 0,25 g), foram mantidos em aqu?rios a uma densidade de 33 camar?es m-2. No primeiro experimento, os camar?es foram alimentados na fase de claro ou na fase de escuro durante 8 dias; no nono dia, os animais foram observados por 15 minutos a cada hora, durante as 12 horas de cada fase do fotoper?odo, sendo registradas as freq??ncias de ocorr?ncia dos comportamentos parado, explora??o, ingest?o de alimento, enterramento, nata??o e rastejamento. Ao final da 12? sess?o de observa??o, foram sacrificados e classificados pelo m?todo da setog?nese, nos est?gios do ciclo de muda A, B, C, D0, D1, D2 ou D3. Como resultado, encontramos que os camar?es em est?gio A apresentam elevada frequencia do comportamento parado. Por outro lado, a freq??ncia de ingest?o de alimento foi muito baixa. Os camar?es em est?gio D3 tamb?m apresentaram baixa ingest?o de alimento e elevada inatividade, associada a elevadas freq??ncias de enterramento. No segundo experimento, os camar?es foram mantidos em aclimata??o fisiol?gica ?s condi??es experimentais por 28 dias, distribu?dos em 12 tratamentos na fase de claro e 12 tratamentos na fase de escuro. Ao final, os animais foram sacrificados e dissecados para avalia??o da atividade de protease espec?fica (tripsina) e atividade de protease n?o espec?fica. De modo geral, esses par?metros n?o variaram entre os animais alimentados na fase de claro e aqueles alimentados na fase de escuro. No entanto, percebemos diferen?as significativas na atividade das proteases espec?fica e n?o espec?fica em rela??o ao tratamento alimentar. Na fase de claro, as maiores atividades proteol?ticas convergiam para a 10? hora ap?s o in?cio da fase de claro, enquanto as menores atividades convergiram para a 6? hora ap?s o in?cio dessa fase. Na fase de escuro, as maiores atividades dessas enzimas convergiram para a 12? hora ap?s o in?cio da fase, enquanto as menores atividades convergiram para a 3? hora ap?s o in?cio da fase. No terceiro experimento, buscamos avaliar as respostas comportamentais dos camar?es a tratamentos alimentares baseados na maior ou menor atividade de enzimas proteol?ticas, considerando os resultados do segundo experimento. As categorias comportamentais observadas foram ?s mesmas do primeiro experimento, com observa??es de 30 minutos (15min antes e 15min depois da oferta alimentar). Encontramos uma varia??o na resposta comportamental em fun??o dos tratamentos, com maior ingest?o de alimento nos camar?es alimentados no hor?rio de maior atividade de enzimas proteol?ticas, da fase de claro. Assim, percebemos que eventos peri?dicos associados ? fisiologia dos camar?es interferem em suas respostas comportamentais, revelando situa??es mais prop?cias ? oferta de alimento, capazes de otimizar o manejo alimentar

Xylella fastidiosa é uma bactéria patogênica encontrada em várias plantas. Esta bactéria secreta proteases extracelulares detectadas em gel de eletroforese, sendo a gelatina usada como substrato co-polimerizado. Três principais bandas protéicas foram detectadas com massa molar (MM) de 122, 84 e 65 kDa produzidas pelo isolado de citros (X0) e duas bandas de aproximadamente 84 e 65 kDa de isolado de videira (9713). Estas bactérias produziram zonas de hidrólise em meio sólido contendo gelatina, caseína e hemoglobina. Os resultados usando a gelatina como substrato foram os melhores para a atividade das proteases. A atividade enzimática das proteases de X. fastidiosa de citros e videira foi completamente inibida por PMSF e parcialmente inibida por EDTA, podendo ser visualizado em gel de eletroforese nativo. A temperatura ótima de atividade protéica foi de 30oC e o pH ótimo de 7,0. Além das proteases secretadas por este fitopatógeno, quitinase e β-1,3-glucanase foram também detectadas no sobrenadante das culturas. Os resultados sugeriram que estas proteases produzidas pela X. fastidiosa de citros e videira pertencem ao grupo das serina e metalo proteases. / Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a co-polymerized substrate. Three major protein bands were produced by strain X0 (citrus) with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. SDS-PAGE activity gel indicated that the protease activities of X. fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30oC with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and β-1,3-glucanase activities were also detected in cultures of X. fastidiosa (citrus). From these results, it is suggested that these proteases produced by strains of X. fastidiosa from citrus and grape, belong to the serine- and metallo-protease group.