A proteolytic activity has been shown to be associated with the heat shock protein 70 (hsp70). In order to study this, I have constructed RNA transcribing vectors with the coding sequences of the D. melanogaster (pBUG7) and the human (pMAN70) genes coding hsp70, and with an internal deletion (pBUG301) in D. melanogaster. Proteins from 37 kDa to 70 kDa were translated in a rabbit reticulocyte lysate in the presence of 35S-methionine from RNA synthesized in vitro off the full length templates (pBUG7, and pMAN70), or altered templates. Restriction digestion of pBUG7 with BamH I and Nar I yields templates that produce carboxy-terminal truncated proteins of 37 kDa and 61 kDa respectively. The full length and the truncated proteins contain a proteolytic activity when assayed by SDS/PAGE in two dimensions. The internally deleted protein does not maintain the proteolytic activity. The proteolytic activity was shown not to be the result of non-enzymatic cleavage. A general serine proteinase inhibitor eliminates the proteolytic activity of the full length human and D. melanogaster hsp70. This evidence shows that the proteolytic activity is directly connected to hsp70.

A heat stable protease was identified as the cause of textural degradation in cooked arrowtooth flounder (Atheresthes stomias) muscle. Maximum proteolytic activity in the fish muscle was observed between 55°C and 60°C and myosin heavy chain appeared to be the primary substrate for the enzyme. Degradation of this myofibrillar protein at 55°C was extremely rapid and myosin heavy chain was completely hydrolyzed to peptide fragments smaller than actin, while actin itself was unaffected. A single strand 32kD proteolytic enzyme was extracted from the muscle and purified 125-fold. The enzyme was stable to freezing for up to 6 months. Activity of the semi-purified enzyme at 55°C was optimal against casein between pH 6.0 and 7.0. Sulfhydryl reagents p-chloromercuriphenylsulfonic acid, iodoacetate, iodoacetamide and cystatin were effective in inhibiting enzyme activity in casein assays. The serine protease inhibitors phenylmethylsulfonylfluoride and trypsin-chymotrypsin inhibitor appeared to activate enzyme activity against casein. Adenosine triphosphate was also an activator. Arrowtooth flounder was then considered as a raw material for surimi, since the surimi process provides for repeated washing of the minced muscle and a final mixing step during which inhibitory substances can be conveniently added. Arrowtooth muscle was monitored at all stages of surimi production. There was no evidence of myosin degradation on sodium dodecyl sulphate polyacrylamide electrophoretic gels at any time during surimi production or during the preparation of samples for testing. However, when the washed mince was incubated at 55°C, 12% residual proteolytic activity was observed. This level was sufficient to degrade the myosin component of surimi gels prepared from the control surimi to which no inhibitors had been added. The food grade substances tested for proteolytic inhibition were bovine blood plasma powder, egg white powder, whey protein concentrate, carrageenan and crude α₂-macroglobulin. Addition of plasma and/or egg white powders to control surimi resulted in a product that was comparable to pollock in functional properties as measured by gel strength, expressible moisture and fold tests. Electrophoretic comparison of surimi made with 1.0% or 2.0% plasma powder or egg white with surimi produced with 0.1% or 0.2% α₂-macroglobulin suggested that the plasma and egg white contributed gel enhancing effects in addition to protease inhibition. Carrageenan was not effective as either a protease inhibitor or gel enhancer. / Graduation date: 1992

Graduation date: 1996

Graduation date: 1994

Cathepsin B was the most active cysteine proteinase in the Pacific whiting (Merluccius productus) fish fillet, and cathepsin L in surimi when the activities of the most active cysteine proteinases (cathepsin L, B, and H) were compared. Cathepsin L showed maximum activity at 55°C in both fish fillet and surimi, indicating its function in myosin degradation during conventional cooking of fish fillet and surimi. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Autolytic analysis of surimi proteins showed that the myosin was the primary target, while actin and myosin light chain showed limited hydrolysis during 2 hr incubation. When purified Pacific whiting proteinase was incubated with various component of fish muscle, proteinase was capable of hydrolyzing purified myofibrils myosin, and native and heat-denatured collagen. The degradation pattern of myofibrils by the proteinase was the same as the autolytic pattern of surimi. Inhibition by the food-grade proteinase inhibitors varied with the catalytic type of proteinase. Beef plasma protein (BPP) had a higher percentage of papain inhibitors, followed by whey protein concentrate (WPC), potato powder (PP), and egg white (EW). On the other hand, EW had a higher percentage of trypsin inhibitors followed by BPP, PP, and WPC. EW inhibited trypsin activity completely at levels as low as 1%. WPC inhibited the autolytic activity of fresh surimi. Bovine serum albumin (BSA) was not effective as WPC. WPC can be used as an inhibitor for the Pacific whiting surimi, but high concentration is required. A limited number of inhibitory components were found, as the components in food-grade inhibitors were characterized by inhibitory activity staining. Both EW and PP showed more serine proteinase inhibitors than cysteine proteinase inhibitors. PP showed one cysteine inhibitory component while EW did not show any. BSA in both WPC and BPP acts as an nonspecific competitive inhibitor and reduces the enzyme activity. An unidentified high molecular weight protein (HMP) found in WPC, BPP, and BSA functions as an alternative substrate for papain while it functions as true inhibitor for trypsin. / Graduation date: 1995

Surimi is a seafood product which is used to manufacture restructured products such as artificial crab and lobster. Surimi is produced from fish fillets by washing to remove sarcoplasmic proteins and increase the concentration of myofibrillar proteins, and mixing with cryoprotectants. A valuable attribute of surimi is its ability to form an elastic gel, the gel network being formed by the myofibrillar proteins of fish muscle. It is generally accepted that the quality of surimi gels is influenced by the activity of endogenous protease which acts on the myofibrillar proteins. The proteases in Pacific whiting surimi (Merluccius productus) are particularly problematic due to their high catalytic activity on muscle myosin. The addition of whey protein concentrate (WPC) to Pacific whiting surimi has been shown to enhance the gel strength of the corresponding products produced from this surimi. The mechanism through which WPC enhances the gel strength of Pacific whiting surimi has not been determined, but it has been suggested that WPC acts to inhibit surimi autoproteolysis. The objective of this study was to determine whether the incorporation of WPC into Pacific whiting surimi inhibits autoproteolysis and/or protects the myosin fraction from proteolytic degradation. The effect of supplementing surimi with WPC, beef plasma protein (BPP) and bovine serum albumin (BSA) on its apparent autoproteolysis activity was determined. Three WPC preparations were tested, WPC 34, 34% protein; WPC 80, 80% protein; and WPC 95, 95% protein. Each of the additives was incorporated at the 1, 2, 3 or 4% level. Proteolysis of surimi and supplemented surimi samples was allowed to occur at 55°C. Proteolytic reaction mixtures were terminated by the addition of trichloroacetic acid (TCA). Proteolytic activity was estimated by measuring the difference in TCA-soluble peptides present in reaction mixtures of paired (identical) samples, one having been incubated at 55°C while the paired sample was kept on ice. Peptides were quantified by the bicinchoninic acid, Lowry, dye-binding and trinitrobenzenesulfonic acid methods. Results based on the different peptide assays were compared in order to asses the reliance of results on specific assay methods. BPP was found to have the most inhibitory activity in the autoproteolysis assays, followed by the WPC preparations and then BSA. Autoproteolysis was completely inhibited by the incorporation of 1% BPP, 3% WPC 80 and 2% WPC 95. The extent of inhibition by the WPC preparations was related to their protein content, the higher the protein content the greater the extent of inhibition per unit weight added to surimi. BSA was not an inhibitor of autoproteolysis under the conditions used in this study. The relative extents of inhibition observed for the different additives were independent of the method used to quantify the soluble peptide products. Each of the additives was also tested for their ability to protect the myosin component of surimi from proteolytic degradation. These experiments were done as described above for the autoproteolysis assays with the exception that following the incubation period a portion of the sample, either surimi or a surimi/additive mixture, was completely solubilized in detergent solution, subjected to SDS-PAGE electrophoresis and visualized by protein staining. In these experiments the additives were incorporated at the 4% level. No apparent degradation of myosin could be detected over a 60 min reaction period for surimi samples that were supplemented with BPP, WPC 80 and WPC 95. In contrast, surimi samples incubated without additive clearly showed a loss of myosin after 15 min reaction period. Some myosin degradation was apparent following the 60 min incubation period for the WPC 34-supplemented surimi. A further experiment was conducted to determine the mechanism through which WPC protects myosin and inhibits autoproteolysis. In this experiment WPC 95 and BPP were separately incubated at 55°C with a crude fish protease preparation, i.e. the reaction mixture approximates that used in the autoproteolysis assays except that it contains no surimi. The results indicate that BPP and WPC 95 behave in a similar manner. However, the results were inconclusive with regard to explaining the additive's mechanism of action. Plausible mechanisms which are consistent with the results are discussed. / Graduation date: 1994

My work concentrates on feeding behavior of overwintering diapausing and post {--} diapausing bark beetles and developmental treshold. This is done either biochemically by measuring the enzymatic activity in the midgut and by assessing the feeding status from the size and consistence of the food bolus in the gut. Detailed knowledge of feeding behaviour and development treshold may help to predict the overwintering success of local populations with all the consequencies for spring dispersal and reproduction.

The diploma thesis is focused on enzyme hydrolysis of potato protein concentrates and fractionation of obtained peptide fragments. Were used protein concentrate from tubers variety Ornella and protein concentrate obtained by swedish company Lyckeby Starch AB. The enzyme hydrolysis lasted 24 hours and were used the proteolytic enzyme alkalasa and trypsin. In this work were prove possitive effect of enzyme hydrolysis on solubility and antioxidative properties of potato protein isolates. The fractionation of obtained peptide hydrolysated was based on systém FPLC (Fast protein liquid chromatography). The fractions contained of peptide fragments about 1, 350 kDa or fragments of smaller moleculary weight. The antixodative activity of subfractions were determIne by method called DPPH. The highest values (2,2 and 2,6 TEAC g/kg) were accured at the subfractions which were separations from Ornella hydrolyzates digeste by enzyme alkalasa.

My work concentrates on feeding behavior of overwintering diapausing and post {--} diapausing bark beetles and developmental treshold. This is done either biochemically by measuring the enzymatic activity in the midgut and by assessing the feeding status from the size and consistence of the food bolus in the gut. Detailed knowledge of feeding behaviour and development treshold may help to predict the overwintering success of local populations with all the consequencies for spring dispersal and reproduction.

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.