In this thesis, optimization of production of lipolytic enzymes by submerzed cultivation of Bacillus subtilis (BS) was studied. Production of lipolytic enzymes was tested in three nutrient media, which differed mainly in main sources of carbon, respectively of nitrogen. The first medium contained mainly extract from calf brain and beef heart (BHIB), the second medium contained peptone and yeast extract (NB) and the third one contained peptone and yeast extract with the addition of 2% (w/v) glucose (NBG). The highest lipolytic activity (0.0784 Uml-1) was measured in NBG medium. Maximum of lipolytic activity was observed before the end of the exponential phase of BS growth in all the media. Temperature optimum in NBG medium was determined from 30 to 50 °C, pH optimum in the range of 5 to 11 and subsequently the temperature stability of lipolytic enzymes produced by the BS was estimated. The activity value was determined spectrometrically using p-nitrophenyllaurate as a substrate. Produced lipolytic enzymes showed maximum activity at 37 °C in the alkaline pH of 8.0. Measurement of temperature stability showed that lipolytic enzymes are relatively thermostable enzymes retaining 100 % of the activity even after 1 hour of cultivation at 30 - 50 °C. The presence of 1% (w/v) olive oil in medium NBG caused a decrease in lipolytic activity by 65 % as well as in pH from 6.5 to 5.4 after 14 days of cultivation. After substitution of glucose by fructose in medium NBG, lipolytic activity showed comparable values during the first week of cultivation. On the other hand, the decrease of lipolytic activity by 29 % in the medium with fructose was observed after 14 days of cultivation. A procedure for the identification of lipolytic enzymes of BS by peptide massfingerprinting was developed to understand the potential of synthetic polyester - poly(e-caprolactone) as a lipase inductor. Degradation study of commercial polyester poly(e-caprolactone) was carried out by submerged cultivation of Bacillus subtilis in NBG medium at initial pH 7.0 and 30 °C for 14 days. PCL (Mn = 10,000, Mw = 14 000) was studied in the form of films (1.0 x 1.0 cm), which were prepared by melt-pressing, rapid cooling of the melt to 4 °C and evaporation of the solvent from 2 % dichlormethane solution. The evaluation of the films shown occurrence of weight loss (7.8 - 17.0 wt.%) together with the formation of numerous holes and cracks in the sample surface in relation to the method of the films preparation. Lipolytic activity values increased by 9 - 17 % in the degradation media compared to control samples. Densitometric monitoring showed also higher increase in cell mass in the degradation medium compared with control samples. Based on the results obtained, the degradation process induced by BS could be suggested.

In dieser Arbeit wird ein Verfahren entwickelt (Infrared Transition Moment Orientational Analysis, IR-TMOA) um die dreidimensionale Ordnung auf molekularer Ebene in infrarot-durchlässigen Systemen zu quantifizieren. Es beruht auf der Messung zahlreicher Infrarotspektren, die unter systematisch variierender Polarisation des einfallenden Lichts und Ausrichtung der Probe relativ zur optischen Achse aufgenommen werden. So wird ein repräsentativer Ausschnitt des dreidimensionalen Absorptionsellipsoids gemessen. Die Tensordarstellung dieses Ellipsoids ist äquivalent zum quadratischen Mittel der Verteilungsfunktion der Orientierung der Übergangsmomente, was wiederum dem Ordnungsparameter entspricht. Von zentraler Bedeutung ist ebenfalls, dass die Ordnung (und Orientierung) spezifisch für verschiedene molekulare Untereinheiten angegeben werden kann, da im mittleren Infrarot die Übergangsdipolmomente definierten, lokalen Schwingungen zugeordnet sind. Im zweiten Teil der Arbeit wird IR-TMOA angewendet um die molekulare Ordnung von verschiedenen amorphen und kristallinen Untereinheiten in teil-kristallinen Polymeren (Polycaprolacton, PCL und Polyethylen, PE) zu bestimmen. So kann der Einfluss der Grenzflächen und der geometrischen Einschränkungen in PCL-Filmen auf Substraten in seiner Temperaturabhängigkeit charakterisiert werden. Ebenso wird erstmalig in freitragenden PCL-Filmen in durch mechanische Streckung plastisch deformierten Bereichen die stark biaxiale molekulare Ordnung quantifiziert. In industriell produzierten PE-Filmen, die unter dem Einfluss von äußerer mechanischer Spannung kristallisieren, wird die biaxiale Ordnung und Orientierung vollständig charakterisiert und in Abhängigkeit der Präparationsbedingungen analysiert. Des weiteren wird die Ordnung in einem System aus zwei Phasen untersucht: einer ferroelektrischen Polymermatrix mit mikrometer-großen Flüssigkristalleinschlüssen (Polymer Dispersed Liquid Crystals). Dies erlaubt es, den Einfluss eines äußeren elektrischen Feldes und des remanenten Feldes der Matrix auf die Flüssigkristalle zu quantifizieren. Durch IR-TMOA wird für alle Systeme, die infrarot aktive Vibrationen aufweisen, eine dreidimensionale molekulare Beschreibung der Orientierung und Ordnung ermöglicht. Dies stellt wiederum einen unverzichtbaren Beitrag zum Verständnis der unterliegenden strukturbildenden Prozesse dar und deren Beitrag zur resultierenden makroskopischen Struktur.

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This Thesis describes the synthesis and characterisation of sulfonamide supported titanium, zirconium and aluminium complexes and their use as ring opening polymerisation catalysts for ε-caprolactone and rac-lactide. Chapter 1 introduces polyester use, development and characterisation in general. Metal catalysed ring opening polymerisation of cyclic esters is considered in a literature review of the field. Titanium, zirconium and aluminium complexes supported by polydentate sulfonamide ligands are also discussed. Chapter 2 describes the synthesis and characterisation of new sulfonamide supported titanium amide, isopropoxide and zirconium isopropoxide complexes. Their application as catalysts for the ring opening polymerisation of ε-caprolactone and rac-lactide is discussed and compared with known zirconium isopropoxide complexes supported by bis(phenolate) amine ligands. Chapter 3 describes the synthesis and characterisation of Cs symmetric titanium amide and alkoxide complexes supported by dianionic, tri- and tetradentate sulfonamide ligands. Zirconium alkyl and amide complexes supported by C3- symmetric trianionic ‘tren’ type ligands bearing three different sulfonamide groups are also presented. The application of these complexes for the ring opening polymerisation of ε-caprolactone and rac-lactide is described and compared with the complexes presented in Chapter 2. Chapter 4 provides an overview of the synthesis and characterisation of aluminium alkoxide and alkyl complexes supported by dianionic, tri- and tetradentate sulfonamide ligands. Solution state behaviour and solid state structures are presented and discussed. An assessment of these complexes for the ring opening polymerisation of rac-lactide is presented. Chapter 5 presents full experimental procedures and characterisation data for the new complexes reported. CD Appendix contains .cif files for all new crystallographically characterised complexes described, and additional polymerisation graphs.

<p>Degradable polymers have gained an increased attention in the field of biomedical applications over the past decades, for example in tissue engineering. One way of improving the biocompatibility of these polymers is by chemical surface modification, however the risk of degradation during the modification procedure is a limiting factor. In some biomedical applications, for example in nerve guides, a patterned surface is desired to improve the cell attachment and proliferation.</p><p>In this thesis a new non-destructive, single-step, and solvent free method for surface modification of degradable polymers is described. Poly(L-lactide) (PLLA) substrates have been functionalized with one of the following vinyl monomers; N-vinylpyrrolidone (VP), acrylamide (AAm), or maleic anhydride (MAH) grafts. The substrates were subjected to a vapor phase atmosphere constituted of a mixture of a vinyl monomer and a photoinitiator (benzophenone) in a closed chamber at very low pressure and under UV irradiation. Poly(ε-caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), and poly(trimethylene carbonate) (PTMC) have been surface modified with VP using the same procedure to show the versatility of the method. The wettability of all of the four substrates increased after grafting. The surface compositions were confirmed by ATR-FTIR and XPS. The VP grafted PLLA, PTMC and PLGA substrates have been shown to be good substrates for the normal human cells i.e. keratinocytes and fibroblasts, to adhere and proliferate on. The topography of substrates with well defined nano patterns was preserved during grafting, since the grafted layer is very thin. We have also shown that the method is useful for a simultaneous chemical and topographical modification of substrates by masked vapor phase grafting. The surface topography was determined with SEM and AFM.</p> / <p>Intresset för användningen av nedbrytbara polymerer till biomedicinska applikationer som till exempel vävnads rekonstruktion har ökat avsevärt de senaste decennierna. Ett sätt att öka biokompatibiliteten hos dessa polymerer är genom kemisk ytmodifiering, men risken för nedbrytning under själva modifieringen är en begränsande faktor. I vissa biomedicinska applikationer, till exempel nervguider, är det önskvärt att ha en väldefinierad ytstruktur för att öka vidhäftningen och tillväxten av celler.</p><p>I den här avhandlingen presenteras en ny ickeförstörande, lösningsmedelsfri enstegsprocess för ytmodifiering av nedbrytbara polymerer. Substrat av poly(L-laktid) (PLLA) har ytfunktionaliserats med var och en av följande vinylmonomerer, N-vinylpyrrolidon (VP), akrylamid (AAm) eller maleinsyraanhydrid (MAH). Substraten har exponerats för en gasfasatmosfär av en blandning av en vinylmonomer och en fotoinitiator (bensofenon) i en tillsluten reaktor vid mycket lågt tryck och under UV-strålning. Metodens mångsidighet har även påvisats genom att ytmodifiera substrat av poly(ε-kaprolakton) (PCL), poly(laktid-co-glykolid) (PLGA) och poly(trimetylen karbonat) (PTMC) med VP. Vätbarheten ökade för alla fyra materialen efter ympning med en vinylmonomer. Ytsammansättningen fastställdes med ATR-FTIR och XPS. De VP ympade filmerna av PLLA, PLGA och PTMC visade sig vara bra substrat för mänskliga celler, i detta fall keratinocyter och fibroblaster, att vidhäfta och växa på. Yttopografin hos filmer med väldefinierade nanomönstrade ytor kunde bevaras efter ympning, tack vare att det ympade lagret är så tunt. Gasfas metoden har också visat sig användbar för att simultant ytmodifiera både kemiskt och topografiskt genom maskad gasfasympning. Yttopografin bestämdes med SEM och AFM.</p>

Degradable polymers have gained an increased attention in the field of biomedical applications over the past decades, for example in tissue engineering. One way of improving the biocompatibility of these polymers is by chemical surface modification, however the risk of degradation during the modification procedure is a limiting factor. In some biomedical applications, for example in nerve guides, a patterned surface is desired to improve the cell attachment and proliferation. In this thesis a new non-destructive, single-step, and solvent free method for surface modification of degradable polymers is described. Poly(L-lactide) (PLLA) substrates have been functionalized with one of the following vinyl monomers; N-vinylpyrrolidone (VP), acrylamide (AAm), or maleic anhydride (MAH) grafts. The substrates were subjected to a vapor phase atmosphere constituted of a mixture of a vinyl monomer and a photoinitiator (benzophenone) in a closed chamber at very low pressure and under UV irradiation. Poly(ε-caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), and poly(trimethylene carbonate) (PTMC) have been surface modified with VP using the same procedure to show the versatility of the method. The wettability of all of the four substrates increased after grafting. The surface compositions were confirmed by ATR-FTIR and XPS. The VP grafted PLLA, PTMC and PLGA substrates have been shown to be good substrates for the normal human cells i.e. keratinocytes and fibroblasts, to adhere and proliferate on. The topography of substrates with well defined nano patterns was preserved during grafting, since the grafted layer is very thin. We have also shown that the method is useful for a simultaneous chemical and topographical modification of substrates by masked vapor phase grafting. The surface topography was determined with SEM and AFM. / Intresset för användningen av nedbrytbara polymerer till biomedicinska applikationer som till exempel vävnads rekonstruktion har ökat avsevärt de senaste decennierna. Ett sätt att öka biokompatibiliteten hos dessa polymerer är genom kemisk ytmodifiering, men risken för nedbrytning under själva modifieringen är en begränsande faktor. I vissa biomedicinska applikationer, till exempel nervguider, är det önskvärt att ha en väldefinierad ytstruktur för att öka vidhäftningen och tillväxten av celler. I den här avhandlingen presenteras en ny ickeförstörande, lösningsmedelsfri enstegsprocess för ytmodifiering av nedbrytbara polymerer. Substrat av poly(L-laktid) (PLLA) har ytfunktionaliserats med var och en av följande vinylmonomerer, N-vinylpyrrolidon (VP), akrylamid (AAm) eller maleinsyraanhydrid (MAH). Substraten har exponerats för en gasfasatmosfär av en blandning av en vinylmonomer och en fotoinitiator (bensofenon) i en tillsluten reaktor vid mycket lågt tryck och under UV-strålning. Metodens mångsidighet har även påvisats genom att ytmodifiera substrat av poly(ε-kaprolakton) (PCL), poly(laktid-co-glykolid) (PLGA) och poly(trimetylen karbonat) (PTMC) med VP. Vätbarheten ökade för alla fyra materialen efter ympning med en vinylmonomer. Ytsammansättningen fastställdes med ATR-FTIR och XPS. De VP ympade filmerna av PLLA, PLGA och PTMC visade sig vara bra substrat för mänskliga celler, i detta fall keratinocyter och fibroblaster, att vidhäfta och växa på. Yttopografin hos filmer med väldefinierade nanomönstrade ytor kunde bevaras efter ympning, tack vare att det ympade lagret är så tunt. Gasfas metoden har också visat sig användbar för att simultant ytmodifiera både kemiskt och topografiskt genom maskad gasfasympning. Yttopografin bestämdes med SEM och AFM. / QC 20101014