Recombinant Candida antarctica lipase B was successfullyproduced in the methylotropic yeast Pichia pastoris. Thespecific activities of Candida antarctica lipase B produced inPichia pastoris and commercial Candida antarctica lipase B fromNovozymes were the same. In shake-flask cultivations theexpression levels were about 25 mg L-1. Production levels couldbe increased to 1.5 g L-1, using a fermentor. A model tosimulate growth and oxygen consumption was described. The highcell density growth could be explained by the low maintenancecoefficient of Pichia pastoris. Enrichment of the aeration withoxygen increased the recombinant protein production. The lipasewas also produced as a fusion to a cellulose binding module.The cellulose binding module did not interfere with thespecific activity of the lipase. With this fusion proteincatalytic reactions can be performed in close proximity to acellulose surface. The binding module can also function as anaffinity tag for purification. Establishment of the Candidaantarctica lipase B production system allowed the engineeringof Candida antarctica lipase B variants. Four differentvariants were produced in order to investigate if electrostaticinteractions contributed to enantioselectivity. Theenantioselectivity of two halogenated secondary alcohols wasdoubled for the Ser47Ala variant. Thisimplied thatelectrostatic interactions are important forenantioselectivity. The Trp104His variant showed a decrease inenantioselectivity for all tested substrates. This was causedby an increase in the size of the stereoselectivity pocket.Symmetrical secondary alcohols of different size were used tomap the stereoselectivity pocket. A substituent as large as apropyl or isopropyl could be accommodated in the pocket of theTrp104His variant. In the wild-type lipase thestereoselectivity pocket was estimated to fit an ethyl group.The enzyme variants were subjected to a thermodynamic study, toelucidate changes in the enthalpic and entropic contributionsto enantioselectivity. The enthalpic and entropic contributionschanged for the different lipase variants and werecompensatory. The compensation was not perfect, allowing forchanges in enantioselectivity. In general one can conclude that rational design of newenzyme properties, in order to change the substrateselectivity, is feasible if based on a thorough model ofsubstrate enzyme interactions. <b>Key words:</b>Protein expression, Candida antarctica lipaseB, Pichia pastoris, sitedirected mutagenesis, fermentation,selectivity

The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved. In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase. In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches. In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance. In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage. / QC20100629

In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which was further shown to be substrate dependent (Paper V). A homology structure of Cv-ωTA guided the creation of an enzyme variant showing reduced isopropyl amine inhibition. / QC20100609

Biocatalysis has become a popular technique in organic synthesis due to high activity and selectivity of enzyme catalyzed reactions. Enantioselectivity is a particularly attractive enzyme property, which is utilized for the production of enantiopure substances. Determination of the temperature dependence of enzyme enantioselectivity allows for thermodynamic analyses that reveal the contribution of differential activation enthalpy, ΔR-SΔH‡, and entropy, ΔR-SΔS‡. In the present investigation the influence of substrate structure, variations on enzyme structure and of reaction media on the enantioselectivity of Candida Antarctica lipase B has been studied. The contribution of enthalpy, ΔR-SΔH‡, and entropy, TΔR-SΔS‡, to the differential free energy, ΔR-SΔG‡, of kinetic resolutions of sec-alcohols were of similar magnitude. Generally the two terms were counteracting, meaning that the enantiomer favored by enthalpy was disfavored by entropy. 3-Hexanol was an exception where the preferred enantiomer was favored both by enthalpy and by entropy. Resolution of 1-bromo-2-butanol revealed non-steric interactions to influence both ΔR-SΔH‡ and ΔR-SΔS‡. Molecular modeling of the spatial freedom of the enzyme-substrate transition state indicated correlation tothe transition state entropy. The acyl chain length was shown to affect enantioselectivity in transesterifications of a sec-alcohol. Point mutations in the active site were found to decrease or increase enantioselectivity. The changes were caused by partly compensatory changes in both ΔR-SΔH‡ and ΔR-SΔS‡. Studies on single and double mutation variants showed that the observed changes were not additive. Enantioselectivity was strongly affected by the reaction media. Transesterifications of a sec-alcohol catalyzed by Candida Antarctica lipase B was studied in eight liquidorganic solvents and supercritical carbon dioxide. A correlation of enantioselectivity and the molecular volume of the solvent was found. Differential activation enthalpy, ΔR-SΔH‡, and entropy, ΔR-SΔS‡, display a compensatory nature. However this compensation is not perfect, which allows for modifications of enantioselectivity. The components of the thermodynamic parameters are highly complex and interdependent but if their roles are elucidated rational design of enantioselective enzymatic processes may be possible. / QC 20100616

Telechelic polyesters have successfully been synthesized with lipase-catalyzed polymerization. The produced telechelics had a high degree of di­functionalization, high purity (requiring little or no workup) and controlled degree of polymerization. The syntheses were performed in one-pot one-step reaction systems. The use of protection/deprotection chemistry was not necessary, since the lipase selectivity was utilized in the syntheses. Two different types of lipase-catalyzed polymerizations were applied – ring-opening polymerization and polycondensation. In ring-opening polymerization telechelics were produced by a combination of initiation, α-functionalization, and linking through termination, w-func­tionalization. In polycondensation different types of end-cappers were used to synthesize telechelics. Several exampels of functional groups were used for end-functionalization - epoxide, methacrylate and tetraallyls. Enzyme kinetic schemes describing the different functionalization met­hods of polyesters are presented and discussed. Stoichiometry and different reaction conditions have been studied to understand the effects these functions have on the final structure of the synthesized telechelics. Polyesters are classified as biodegradable, and can also be synthesized from materials that can be extracted or fermented from renewable sources like plants. Lipase-catalysts have several beneficial attributes, like high selectivity, they are renewable and biodegradable, are non-toxic and metal-free and can operate under mild reaction conditions. The focus of this thesis has been on lipase-catalyzed syntheses and characterization of the produced telechelics, in addition some materials have been produced. Some uses of telechelics are surface modification, materials for block co-polymers, functional films and biomedical applications. / QC20100726

<p>A metagenomic gene library prepared from soil found beneath a mummified seal carcass in the Miers Valley, Antarctica, suggests an environment rich in uncharacterised biodiversity including enzymes with possible application to industrial processes. A sequence based gene mining investigation was performed on a clone, which archives a metagenomic sequence from this environment. The sequence was annotated using de novo bioinformatics and molecular biology techniques. A predicted NAD+-dependent DNA ligase, ligDB1 was selected for further characterisation. LigDB1 encodes a gene product that contains all the sequence features of a functional ligase. The protein was overexpressed in a heterologous E. coli host and purified to homogeneity. LigDB1 did not exhibit nick sealing activity, but was able to perform AMP-dependent DNA relaxation in the presence of high concentrations of enzyme. DNA modifying enzymes from cold environments perform optimally at low temperatures and may be of use as molecular tools in biotechnology. Complete characterisation of this enzyme is subject to further investigations.</p>

Anjoner från de tre korta organiska syrorna: acetat, format och MSA är intressanta att mäta då de kan användas för olika sorters miljöstudier. Jonkromatografen vid Institutionen för Geovetenskaper kan för närvarande inte skilja på dessa tre ämnen, därför utvecklades sex nya metoder i detta projekt för att lösa problemet. Metod 5 visade sig vara den bästa, där acetat och format separerades. Resultatet ansågs vara bra, även om MSA inte separerades. Metod 5 testades sedan på ett par naturliga prover; vatten-, snö- och isprover. Alla dessa prover visade en större mängd av format än acetat, som i vissa fall inte ens visades. Resultaten verkade rimliga, inte många av dem stack ut i jämförelse till andra resultat av samma fas. Det ytligaste provet från Lomonosovfonnaglaciären skiljer sig dock ganska mycket i mängden format jämfört med prover från andra djup av denna iskärna, förmodligen på grund av kontamination vid hanteringen av proverna både vid provtagningen och i labbet. MSA kan dock även mätas om man vet mängden acetat och format i provet. Detta görs genom tillsats av kända mängder av MSA till samma prov i efterföljande körningar, för att sedan kunna beräkna koncentrationen av MSA i provet. Problemet med denna metod är att koncentrationen av MSA måste vara tillräckligt hög i kontrast till koncentrationerna av acetat och format för att få tillförlitliga resultat, vilket den inte var i det här projektets analyserade prover. / Anions from three short organic acids: acetate, formate and MSA are interesting to measure since they can be used for different environmental studies. The ion-chromatographer at the Department of Earth Sciences is currently not able to separate these three substances; therefore six new methods were developed in this project to solve this problem. Short organic test 5 ended up to be the best method, where acetate and formate were separated. The result was considered good, even if MSA were not separated. Method 5 was then tested on a couple of natural water, snow and ice samples. All these samples showed a larger amount of formate than of acetate, which in some cases was not even found. The results seemed plausible; not many of them were sticking out compared to others of the same phase. The shallowest sample from the Lomonosovfonna ice cap did however differ quite a lot in amount of formate compared with samples from other depths of this ice core; probably because of contamination which could have occurred at both the ice cap and in the lab during the handling of the samples. MSA can however also be measured if the amount of acetate and formate in the sample is known. This is done by adding known amounts of MSA to the same sample in subsequent runs to then be able to calculate the concentration of MSA in the sample. The problem with the use of this method is that the concentration of MSA needs to be high enough in contrast to acetate and formate in order to get reliable results, which was not the case in the samples measured in this project.

Abstract Methods for tephrochronology are evaluated, in the following way: Lake sediments &lt;500 years old from three small Antarctic lakes were analysed for identification of tephras. Subsamples were analysed for a) grain size, and identification and concentration of volcanogenic grains, b) identification of tephra horizons, c) element abundance by EPMA WDS/EDS and LA-ICP-MS, and d) possible correlations between lakes and volcanoes. Volcanogenic minerals and shards were found all through the sediment cores in all three lakes, in different abundances. A high background population of volcanogenic mineral grains, in all samples, made the identification of tephra horizons difficult, and shards could only be distinguished by certainty after chemical analysis of elements. The tephra layers commonly could not be seen by the naked eye, and, hence they are regarded as cryptotephras. Because of the small size of recent eruptions in the research area, and the travel distance of ash, most shards are small and difficult to analyse. Nine possible tephra horizons have been recorded in the three lakes, and preliminary correlations have been made. But because of analytical problems, the proposed correlations between the lakes and possible volcanic sources are preliminary.

Die geodätischen Beobachtungsverfahren leisten auf verschiedene Weise Beiträge zur Erforschung des Systems Erde: Einerseits beobachten sie die rezenten Prozesse und ihre zeitlichen Variationen direkt, andererseit liefert sie die Grundlage für die konsistente Betrachtung aller Einflüsse in einem einheitlichen geometrischen und gravimetrischen Bezug. Das Projekt des Global Geodetic Observing System (GGOS) der Internationalen Assoziation für Geodäsie (IAG) soll die Voraussetzungen zur Vereinigung der verschiedenen geodätischen Beobachtungsverfahren, Modelle und Auswertemethoden mit dem Ziel schaffen, mit einem konsistenten Satz geodätischer Parameter ein hochgenaues Monitoring des Systems Erde zu ermöglichen. Die Realisierung geodätischer Bezugssysteme mit höchsten Genauigkeitsansprüchen ist in diesem Kontext eine zentrale Aufgabe des GGOS und Thema der vorliegenden Arbeit. In der derzeit üblichen Darstellung umfasst eine Realisierung des Terrestrischen Referenzsystems (TRS) Stationspositionen zu einer spezifischen Epoche und ihre linearen Änderungen mit der Zeit. In diesem Konzept führen alle nichtlinearen Stationsbewegungen zu residualen Abweichungen, die geowissenschaftlich interpretiert werden können. Der natürliche Ursprung eines globalen TRS, so auch des International Terrestrial Reference System (ITRS), liegt im Massezentrum des Systems Erde (CM). Mit Hilfe dynamischer Satellitenverfahren, wie GPS, lässt sich dieser Ursprung aus geodätischen Beobachtungen realisieren. In einem konsistenten Ausgleichungsansatz werden Satellitenbahnen, Stationspositionen und die in Kugelflächenfunktionen niedrigen Grades modellierte Auflastdeformation gemeinsam geschätzt. Die Grundlage der Realisierung des ITRS bilden in einem gemeinsamen Projekt der TU Dresden, der TU München und des GFZ Potsdam reprozessierte Beobachtungen eines über 200 Stationen umfassenden globalen GPS-Netzes des Beobachtungszeitraums 1994 bis 2007. Nach der Vorstellung der Grundprinzipien des GPS und seiner wesentlichen Fehlereinflüsse erfolgt die Beschreibung der Analyse der Beobachtungsdaten selbst. Sie umfasst die einheitliche Auswertung über den gesamten Zeitraum sowie Verbesserungen in der Modellierung der atmosphärischen Einflüsse und der Charakteristika der Sende- und Empfangsantennen sowie die Nutzung der Normalgleichungen zu Realisierung des ITRS. Der abgeleitete Terrestrische Referenzrahmen (TRF) wird Potsdam-Dresden-Reprocessing 2007 (PDR07) genannt. Zur Beurteilung der Genauigkeit und Zuverlässigkeit dieses TRF werden umfangreiche Analysen durchgeführt. So wird der PDR07 u.a. mit weiteren Realisierungen des ITRS, dem ITRF2000, dem ITRF2005 und den Realisierungen des International GNSS Service (IGS) IGb00 und IGS05, verglichen. Für eine Vielzahl geodynamischer Anwendungen werden GPS-Stationen in Messkampagnen beobachtet. Die hochgenaue Realisierung des ITRS in diesen regionalen GPS-Netzen ist für die geodynamische Interpretation der Ergebnisse zwingend erforderlich. Am Beispiel eines regionalen GPS-Netzes in der Antarktis wird untersucht, wie sich das ITRS in derartigen Netzen realisieren lässt und mit welcher Genauigkeit lineare Stationsbewegungen aus Kampagnenmessungen abgeleitet werden können. Im Anschluss werden die erhaltenen Bewegungsraten geodynamisch interpretiert: Aus den horizontalen Bewegungsraten wird die Bewegung der Antarktischen Kontinentalplatte im Konzept der Globaltektonik bestimmt und ihre innere Stabilität bewertet. Die vertikalen Stationsbewegungen werden genutzt, um Aussagen über rezente Krustendeformationen aufgrund glazialisostatischer Ausgleichsbewegungen und rezenter Massenvariationen des antarktischen Eises zu treffen. / The geodetic observation techniques contribute in several ways to the research of the system Earth: On the one hand they observe the recent processes and their variations in time directly, on the other hand they provide the basis for a consistent description of all effects in a consistent geometrical and gravimetrical reference. Within the project Global Geodetic Observing System (GGOS) of the International Association of Geodesy (IAG) the prerequisites for the combination of geodetic observation techniques, models and analysis strategies shall be created in order to enable a high accurate monitoring of the system Earth with consistent geodetic parameters. In this context the realization of geodetic reference systems with highest accuracy is a central task of the GGOS and subject of this thesis. At present, a common realization of the Terrestrial Reference System (TRS) consists of station positions according to a specific epoch and their linear changes with time. In this concept non-linear station motions yield to residual variations, which may be used for geoscientific interpretations. The natural origin of a global TRS, and this is also the case for the International Terrestrial Reference System (ITRS), is the center of mass of the system Earth (CM). This origin can be realized by observations of dynamic satellite techniques, such as GPS. In a consistent approach satellite orbits, stations positions and the lower degrees of harmonic surface mass load coefficients are estimated simultaneously. The ITRS is realized based on reprocessed observations of a global GPS network. In a joint effort TU Dresden, TU München and GFZ Potsdam analyzed the data of more than 200 stations of the observation time span 1994 to 2007. After an introduction to the basic principles of GPS and its major error sources the data analysis is described. This covers a homogeneous analysis over the entire period, improvements in atmosphere modeling and antenna phase center modeling as well as the usage of normal equations for the ITRS realization. The determined Terrestrial Reference Frame (TRF) is named Potsdam-Dresden-Reprocessing 2007 (PDR07). In order to assess the accuracy and stability of this TRF a variety of analyses is performed. For example, PDR07 is compared to other ITRS realizations, such as the ITRF2000, the ITRF2005 as well as the realizations of the International GNSS Service (IGS) IGb00 and IGS05. GPS campaign observations are often used to investigate geodynamic phenomena. The realization of the ITRS with highest accuracy in these regional GPS networks is essential for the geodynamic interpretation of the results. A regional GPS network in Antarctica is used to investigate the optimal way to realize the ITRS in such networks and the accuracy of linear station rates determined from campaign observations. Subsequently, the station rates are used for geodynamic interpretations: The horizontal station rates are used to determine the movement of the Antarctic Plate in the concept of global plate kinematics and to assess the inner stability of the Antarctic Plate. The vertical station rates are used to evaluate recent crustal deformations caused by glacial isostatic adjustment and recent mass changes of the Antarctic ice sheet.

<p>Recombinant Candida antarctica lipase B was successfullyproduced in the methylotropic yeast Pichia pastoris. Thespecific activities of Candida antarctica lipase B produced inPichia pastoris and commercial Candida antarctica lipase B fromNovozymes were the same. In shake-flask cultivations theexpression levels were about 25 mg L-1. Production levels couldbe increased to 1.5 g L-1, using a fermentor. A model tosimulate growth and oxygen consumption was described. The highcell density growth could be explained by the low maintenancecoefficient of Pichia pastoris. Enrichment of the aeration withoxygen increased the recombinant protein production. The lipasewas also produced as a fusion to a cellulose binding module.The cellulose binding module did not interfere with thespecific activity of the lipase. With this fusion proteincatalytic reactions can be performed in close proximity to acellulose surface. The binding module can also function as anaffinity tag for purification. Establishment of the Candidaantarctica lipase B production system allowed the engineeringof Candida antarctica lipase B variants. Four differentvariants were produced in order to investigate if electrostaticinteractions contributed to enantioselectivity. Theenantioselectivity of two halogenated secondary alcohols wasdoubled for the Ser47Ala variant. Thisimplied thatelectrostatic interactions are important forenantioselectivity. The Trp104His variant showed a decrease inenantioselectivity for all tested substrates. This was causedby an increase in the size of the stereoselectivity pocket.Symmetrical secondary alcohols of different size were used tomap the stereoselectivity pocket. A substituent as large as apropyl or isopropyl could be accommodated in the pocket of theTrp104His variant. In the wild-type lipase thestereoselectivity pocket was estimated to fit an ethyl group.The enzyme variants were subjected to a thermodynamic study, toelucidate changes in the enthalpic and entropic contributionsto enantioselectivity. The enthalpic and entropic contributionschanged for the different lipase variants and werecompensatory. The compensation was not perfect, allowing forchanges in enantioselectivity.</p><p>In general one can conclude that rational design of newenzyme properties, in order to change the substrateselectivity, is feasible if based on a thorough model ofsubstrate enzyme interactions.</p><p><b>Key words:</b>Protein expression, Candida antarctica lipaseB, Pichia pastoris, sitedirected mutagenesis, fermentation,selectivity</p>