<p>The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity (&gt / 1%) was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study.</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. 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

During attempted enzyme-catalysed resolution of sterically hindered secondary alcohols, hemiacetals and their esters were unexpectedly detected. Hemiacetal esters are reactive compounds that decompose to alcohol, aldehyde and acid under ordinary work-up conditions i.e. in contact with water, acid, or silica gel. Thus, the presence of these side products might decrease the enantiomeric excess of the residual alcohol after workup of a lipase-catalysed resolution. The formation of these hemiacetal esters were further studied using both terpenoid and non-terpenoid substrate alcohols, various acyl donors, and lipases. The prerequisite for their formation is the presence of a sterically hindered substrate alcohol, an aldehyde or an aldehyde releasing acyl donor, and a lipase (PCL-L6, PCL-PS and CAL-B). Enantioselective synthesis of (S)- and (R)-cryptone was performed via a ring closing metathesis (RCM) of (S)- and (R)-6-isopropyl-1,7-octadien-3-one. The stereochemistry was induced by using pseudoephedrine as chiral auxiliary in an alkylation reaction which provided a chiral octadienone. Problems with removal of the RCM-catalyst resulted in low yields and low enantiomeric purity. In an alternative approach, racemic cryptone was subjected to conjugate addition with thiophenol followed by reduction to the corresponding alcohol. Lipase-catalysed resolution of this alcohol yielded, after oxidation and elimination, (R)- and (S)- cryptone with 76% and 98% ee, respectively. Marine fouling of immersed objects is a serious problem. Many coatings contain effective antifouling compounds having the drawback of being toxic to the marine environment. The marine natural product 10-isocyano-4-cadinene is a potentially non-toxic antifouling agent against the barnacle Balanus amphitrite and therefore an interesting target for organic synthesis. Cryptone was used as a starting material in attempted syntheses of this compound and other similar model compounds. / QC 20100901

Enzymes as tools in organic synthesis have provided enormous advantages. This thesis deals with the applications of enzymes in the kinetic resolutions of racemic compounds. The stereochemistry of chiral compounds and the kinetics of α/β hydrolase lipases are presented. From a practical point of view, the handling of a large number of parameters that influences the kinetic resolutions, especially enantioselectivity (E-value) are systematically described. A variety of approaches employed for raising the yields to over 50% are additionally discussed. Methods for the preparation of synthetically useful chiral building blocks were developed in this thesis. Thus, resolution of secondary alcohols bearing two vicinal stereocentres are studied. These building blocks can serve as starting materials for the synthesis of various enantiomerically pure compounds for agrochemistry, pharmaceuticals, chemical industry, and particularly for the total synthesis of pheromones. Racemic 3-substitued 2-hydroxybutane derivatives were produced in fairly high diastereomeric purities by a variety of chemical approaches, such as epimerization, metal-catalysed asymmetric addition etc. Kinetic resolution of these racemates was achieved by enzyme-catalysed reactions. Two lipases, Candida antarctica lipase B and Pseudomonas cepacia lipase were found to be useful in acylations as well as hydrolyses. In the biotransformations studied, the presence and nature of the second vicinal stereocentre in the chiral secondary alcohols investigated seemed to be important, e.g. in terms of the efficiencies of sequential kinetic resolutions, and altering the selectivities as well. / QC 20101020

Enzymes are gaining increasing importance as catalysts for selective transformations in organic synthetic chemistry. The engineering and design of enzymes is a developing, growing research field that is employed in biocatalysis. In the present thesis, combinatorial protein engineering methods are applied for the development of Candida antarctica lipase A (CALA) variants with broader substrate scope and increased enantioselectivity. Initially, the structure of CALA was deduced by manual modelling and later the structure was established by X-ray crystallography. The elucidation of the structure of CALA revealed several biocatalytically interesting features. With the knowledge derived from the enzyme structure, enzyme variants were produced via iterative saturation mutagenesis (ISM), a powerful protein engineering approach. Several of these variants were highly active and enantioselective towards bulky esters. Furthermore, an extensively combinatorial protein engineering approach was developed and investigated. A CALA variant with a spacious substrate binding pocket that can accommodate an unusually bulky substrate, an ester derivate of the non-steroidal anti-inflammatory drug (S)-ibuprofen, was obtained with this approach. / At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr. 5: Manuscript

In the early nineties cannabinoid receptors, the main target for Δ9-tetrahydrocannabinol (THC), the psychoactive component of marijuana were identified. Shortly after their endogenous ligands, N-arachidonoylethanolamine (anandamide, AEA) and 2-diacylglycerol (2-AG) were characterized. The enzymes primarily responsible for catalysing the degradation of AEA and 2-AG are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL) respectively. AEA is a member of the N-acylethanolamine (NAE) class of lipids, which depending on the acyl chain length and number of double bonds can act as ligands for a variety of biological targets. Exogenous cannabinoids have long been reported to have analgesic effects, however the clinical usefulness of such substances is limited by their psychoactive effects. Inhibition of endocannabinoid degradation would mean enhancing the therapeutic effects without producing these unwanted side effects. In order to succeed in developing such compounds the pharmacology of the enzymes responsible for the degradation of endocannabinoids has to be thoroughly understood. When the preclinical part of this thesis was planned, FAAH had been well characterized whereas little was known as to the pharmacology of MGL. A series of compounds were tested in this first study aiming to find MGL-selective compounds. Although no compounds showed selectivity for MGL over FAAH, several interesting agents affecting both enzymes were identified. In order to increase the knowledge concerning which patient group would benefit from such treatment strategies it is important to investigate in which pain states the endocannabinoids/NAEs are altered. Thus the general aim of the clinical part of this thesis was to investigate the levels of endocannabinoids/NAEs in the interstitium of the trapezius muscle in women suffering from chronic neck/shoulder pain (CNSP) and chronic wide spread pain (CWP) and in healthy pain-free controls. Furthermore for the CNSP the effect of training, which is a commonly recommended treatment for these patients, on the levels of endocannabinoids/NAEs was also investigated. Microdialysis technique in the trapezius muscle was used for sampling and masspectrometry was used for analysing. Two NAEs, N-palmitoylethanolamine (PEA) and N-stearoylethanolamine (SEA), could be repeatedly measured. The levels of these two lipids were significantly higher in CNSP compared to CON. The result showed also that PEA and SEA mobilize differently in CWP compared to both CNSP and CON. Taken together the results presented in thesis represent an early characterization of the pharmacology of MGL and provides novel information on NAEs in chronic muscle pain.

L'hépatite C reste un problème majeur de santé publique. Malgré la mise au point d'un modèle de réplication du virus de l'hépatite C (VHC) in vitro, les mécanismes conduisant à l'infection restent encore mal connus. Le VHC est sécrété et circule dans le sérum associé à des lipoprotéines. L'importance des lipoprotéines pour le cycle viral nous a conduits à étudier le rôle de la lipoprotéine lipase (LPL), une enzyme lipolytique, dans l'infection de la cellule par le VHC. Nous avons montré que la LPL potentialise l'attachement et l'internalisation du virus par un mécanisme similaire à la clearance hépatique des lipoprotéines. La LPL dimérique forme un pont entre les lipoprotéines associées au virus et les HSPG à la surface des cellules. Néanmoins son action conduit à une inhibition de l'infection par les souches virales JFH-1 et J6/JFH-1 produites en culture cellulaire et dans les hépatocytes humains greffés à des souris chimériques uPA-SCID. L'analyse par ultracentrifugation en gradient d'iodixanol des virus produits in vitro et in vivo a montré la présence de deux populations virales : la première, de densité très faible, est beaucoup plus infectieuse que la seconde, de densité plus élevée. L'infection in vitro par ces deux populations virales est inhibée par la LPL. La LPL représente donc un nouvel inhibiteur de l'infection par le VHC. Nous avons également démontré que la présence d'un réseau de microtubules intact et dynamique est cruciale pour l'entrée du VHC et les étapes post-fusion qui mènent à l'infection. Enfin, nous avons mis en évidence une interaction de la protéine de capside avec les tubulines α et β, conduisant à une augmentation de la polymérisation des microtubules. Ces observations suggèrent que le VHC pourrait utiliser les mécanismes de polymérisation des microtubules pour établir l'infection, et la protéine de capside jouer un rôle essentiel dans ce processus. Les nouvelles approches antivirales pourraient donc cibler les éléments du cytosquelette et/ou des lipoprotéines associées aux particules virales.