Biochemical Modification of Thermomechanical Pulp Fibers

The first objective of this study was to find out reliable laboratory methods to predict the effect of enzymes on specific energy consumption and fiber properties of TMP pulp. The second one was to find with interactive software called “Knowledge discovery in databases” enzymes or other additives that can be used in finding a solution to reduce energy consumption of TMP pulp. The chemical composition of wood and enzymes, which have activity on main wood components were presented in the literature part of the work. The results of previous research in energy reduction of TMP process with enzymes were also highlighted. The main principles of knowledge discovery have been included in literature part too. The experimental part of the work contains the methods description in which the standard size chip, crushed chip and fiberized spruce chip (fiberized pulp) were used. Different types of enzymatic treatment with different dosages and time were tested during the experiments and showed. Pectinase, endoglucanase and mixture of enzymes were used for evaluation of method reliability. The fines content and fiber length of pulp was measured and used as evidence of enzymes' effect. The refining method with “Bauer” laboratory disc refiner was evaluated as not highly reliable. It was not able to provide high repeatability of results, because of uncontrolled feeding capacity and refining consistency. The refining method with Valley refiner did not have a lot of variables and showed stable and repeatable results in energy saving. The results of experiments showed that efficient enzymes impregnation is probably the main target with enzymes application for energy saving. During the work the fiberized pulp showed high accessibility to enzymatic treatment and liquid penetration without special impregnating equipment. The reason was that fiberized pulp has larger wood surface area and thereby the contact area between the enzymatic solution and wood is also larger. Standard size chip and crushed chip treatment without special impregnator of enzymatic solution was evaluated as not efficient and did not show visible, repeatable results in energy consumption decrease. Thereby it was concluded that using of fiberized pulp and Valley refiner for measurements of enzymes' effectiveness in SEC decrease is more suitable than normal size chip and crushed chip with “Bauer” refiner. Endoglucanase with 5 kg/t dosage showed about 20% energy consumption decrease. Mixture of enzymes with 1.5 kg/t dosage showed about 15% decrease of energy consumption during the refining. Pectinase at different dosages and treatment times did not show significant effect on energy consumption. Results of knowledge discovery in databases showed the xylanase, cellulase and pectinase blend as most promising for energy reduction in TMP process. Surfactants were determined as effective additives for energy saving with enzymes.

NADPH-Dependent Thioredoxin System In Regulation of Chloroplast Functions

Photosynthesis, the process in which carbon dioxide is converted into sugars using the energy of sunlight, is vital for heterotrophic life on Earth. In plants, photosynthesis takes place in specific organelles called chloroplasts. During chloroplast biogenesis, light is a prerequisite for the development of functional photosynthetic structures. In addition to photosynthesis, a number of other metabolic processes such as nitrogen assimilation, the biosynthesis of fatty acids, amino acids, vitamins, and hormones are localized to plant chloroplasts. The biosynthetic pathways in chloroplasts are tightly regulated, and especially the reduction/oxidation (redox) signals play important roles in controlling many developmental and metabolic processes in chloroplasts. Thioredoxins are universal regulatory proteins that mediate redox signals in chloroplasts. They are able to modify the structure and function of their target proteins by reduction of disulfide bonds. Oxidized thioredoxins are restored via the action of thioredoxin reductases. Two thioredoxin reductase systems exist in plant chloroplasts, the NADPHdependent thioredoxin reductase C (NTRC) and ferredoxin-thioredoxin reductase (FTR). The ferredoxin-thioredoxin system that is linked to photosynthetic light reactions is involved in light-activation of chloroplast proteins. NADPH can be produced via both the photosynthetic electron transfer reactions in light, and in darkness via the pentose phosphate pathway. These different pathways of NADPH production enable the regulation of diverse metabolic pathways in chloroplasts by the NADPH-dependent thioredoxin system. In this thesis, the role of NADPH-dependent thioredoxin system in the redox-control of chloroplast development and metabolism was studied by characterization of Arabidopsis thaliana T-DNA insertion lines of NTRC gene (ntrc) and by identification of chloroplast proteins regulated by NTRC. The ntrc plants showed the strongest visible phenotypes when grown under short 8-h photoperiod. This indicates that i) chloroplast NADPH-dependent thioredoxin system is non-redundant to ferredoxinthioredoxin system and that ii) NTRC particularly controls the chloroplast processes that are easily imbalanced in daily light/dark rhythms with short day and long night. I identified four processes and the redox-regulated proteins therein that are potentially regulated by NTRC; i) chloroplast development, ii) starch biosynthesis, iii) aromatic amino acid biosynthesis and iv) detoxification of H₂O₂. Such regulation can be achieved directly by modulating the redox state of intramolecular or intermolecular disulfide bridges of enzymes, or by protecting enzymes from oxidation in conjunction with 2-cysteine peroxiredoxins. This thesis work also demonstrated that the enzymatic antioxidant systems in chloroplasts, ascorbate peroxidases, superoxide dismutase and NTRC-dependent 2-cysteine peroxiredoxins are tightly linked up to prevent the detrimental accumulation of reactive oxygen species in plants.

Astringent Food Compounds and Their Interactions with Taste Properties

Astringency is traditionally thought to be induced by plant tannins in foods. Because of this current research concerning the mechanism of astringency is focused on tannin‐protein interactions and thus on precipitation, which may be perceived by mechanoreceptors. However, astringency is elicited by a wide range of different phenolic compounds, as well as, some non‐phenolic compounds in various foods. Many ellagitannins or smaller compounds that contribute to astringent properties do not interact with salivary proteins and may be directly perceived through some receptors. Generally, the higher degree of polymerization of proanthocyanidins can be associated with more intense astringency. However, the astringent properties of smaller phenolic compounds may not be directly predicted from the structure of a compound, although glycosylation has a significant role. The astringency of organic acids may be directly linked to the perception of sourness, and this increases along with decreasing pH. Astringency can be divided into different sub‐qualities, including even other qualities than traditional mouth‐drying, puckering or roughing sensations. Astringency is often accompanied by bitter or sour or both taste properties. The different sub‐qualities can be influenced by different astringent compounds. In general, the glycolysation of the phenolic compound results in more velvety and smooth mouthdrying astringency. Flavonol glycosides and other flavonoid compounds and ellagitannins contribute to this velvety mouthdrying astringency. Additionally, they often lack the bitter properties. Proanthocyanidins and phenolic acids elicit more puckering and roughing astringency with some additional bitter properties. Quercetin 3‐O‐rutinoside, along with other quercetin glycosides, is among the key astringent compounds in black tea and red currants. In foods, there are always various other additional attributes that are perceived at the same with astringency. Astringent compounds themselves may have other sensory characteristics, such as bitter or sour properties, or they may enhance or suppress other sensory properties. Components contributing to these other properties, such as sugars, may also have similar effects on astringent sensations. Food components eliciting sweetness or fattiness or some polymeric polysaccharides can be used to mask astringent subqualities. Astringency can generally be referred to as a negative contributor to the liking of various foods. On the other hand, perceptions of astringent properties can vary among individuals. Many genetic factors that influence perceptions of taste properties, such as variations in perceiving a bitter taste or variations in saliva, may also effect the perception of astringency. Individuals who are more sensitive to different sensations may notice the differences between astringent properties more clearly. This may not have effects on the overall perception of astringency. However, in many cases, the liking of astringent foods may need to be learned by repetitive exposure. Astringency is often among the key sensory properties forming the unique overall flavour of certain foods, and therefore it also influences whether or not a food is liked. In many cases, astringency may be an important sub‐property suppressed by other more abundant sensory properties, but it may still have a significant contribution to the overall flavour and thus consumer preferences. The results of the practical work of this thesis show that the astringent phenolic compounds are mostly located in the skin fractions of black currants, crowberries and bilberries (publications I–III). The skin fractions themselves are rather tasteless. However, the astringent phenolic compounds can be efficiently removed from these skin fractions by consecutive ethanol extractions. Berries contain a wide range of different flavonol glycosides, hydroxycinnamic acid derivatives and anthocyanins and some of them strongly contribute to the different astringent and bitterness properties. Sweetness and sourness are located in the juice fractions along with the majority of sugars and fruit acids. The sweet and sour properties of the juice may be used to mask the astringent and bitterness properties of the extracts. Enzymatic treatments increase the astringent properties and fermented flavour of the black currant juice and decrease sweetness and freshness due to the effects on chemical compositions (IV). Sourness and sweetness are positive contributors to the liking of crowberry and bilberry fractions, whereas bitterness is more negative (V). Some astringent properties in berries are clearly negative factors, whereas some may be more positive. The liking of berries is strongly influenced by various consumer background factors, such as motives and health concerns. The liking of berries and berry fractions may also be affected by genetic factors, such as variations in the gene hTAS2R38, which codes bitter taste receptors (V).

Runkopaperin imukyvyn optimointi

Diplomityön tarkoituksena oli optimoida laminaatin valmistuksessa käytettävän runkopaperin imukyky niin, että pienemmällä hartsimäärällä saavutetaan vähintään jo olemassa olevat impregointi- ja laminaatin ominaisuudet tai että nykyisellä hartsimäärällä saadaan nopeampi imeytyminen läpi paperirakenteen. Kirjallisuusosassa etsittiin ja selvitettiin erilaisten tekijöiden tai toimintatapojen vaikutukset, joilla voitaisiin muokata paperin imukyky halutulle tasolle. Kirjallisuudesta löydetyistä tekijöistä ja menetelmistä valittiin kokeelliseen osaan tutkittavaksi potentiaalisimmat sekä toteutuskelpoisimmat tavat. Diplomityön kokeellinen osuus koostui kahdesta osasta. Ensimmäisessä vaiheessa kirjallisuudesta löydettyjä menettelyjä testattiin laboratoriomittakaavassa erilaisilla esikokeilla. Työn toisessa vaiheessa suoritettiin pilotpaperikoneella koeajo, jossa tutkittiin tarkemmin esikokeiden menetelmiä, jotka olivat antaneet lupaavia tuloksia. Lisäksi tutkittiin menetelmiä, joita ei esikokeissa kokeiltu, mutta jotka olivat teoreettisesti kiinnostavia. Tulosten perusteella paperin hartsinottokykyä pystyttiin madaltamaan nestemäisen AKD-liiman sekä erään kemikaalin avulla. Hartsin imeytymisnopeutta paperiin pystyttiin kasvattamaan kuidun entsymaattisella käsittelyllä, lämpösarveistamalla sekä käyttämällä erästä pinta-aktiivista ainetta.

In vivo imaging of vascular adhesion protein 1 - preclinical studies with positron emission tomography

The golden standard in nuclear medicine imaging of inflammation is the use of radiolabeled leukocytes. Although their diagnostic accuracy is good, the preparation of the leukocytes is both laborious and potentially hazardous for laboratory personnel. Molecules involved in leukocyte migration could serve as targets for the development of inflammation imaging agents. An excellent target would be a molecule that is absent or expressed at low level in normal tissues, but is induced or up-regulated at the site of inflammation. Vascular adhesion protein-1 (VAP-1) is a very promising target for in vivo imaging, since it is translocated to the endothelial cell surface when inflammation occurs. VAP-1 functions as an endothelial adhesion molecule that participates in leukocyte recruitment to inflamed tissues. Besides being an adhesion molecule, VAP-1 also has enzymatic activity. In this thesis, the targeting of VAP-1 was studied by using Gallium-68 (68Ga) labeled peptides and an Iodine-124 (124I) labeled antibody. The peptides were designed based on molecular modelling and phage display library searches. The new imaging agents were preclinically tested in vitro, as well as in vivo in animal models. The most promising imaging agent appeared to be a peptide belonging to the VAP-1 leukocyte ligand, Siglec-9 peptide. The 68Ga-labeled Siglec-9 peptide was able to detect VAP-1 positive vasculature in rodent models of sterile skin inflammation and melanoma by positron emission tomography. In addition to peptides, the 124I-labeled antibody showed VAP-1 specific binding both in vitro and in vivo. However, the estimated human radiation dose was rather high, and thus further preclinical studies in disease models are needed to clarify the value of this imaging agent. Detection of VAP-1 on endothelium was demonstrated in these studies and this imaging approach could be used in the diagnosis of inflammatory conditions as well as melanoma. These studies provide a proof-of-concept for PET imaging of VAP-1 and further studies are warranted.

Purified lignin for demanding applications

Diplomityön tarkoituksena oli puhdistaa kraft ligniiniä. Raaka-aineena käytettiin pääasiassa Lignoboost -menetelmän kaltaisella menetelmällä havupuu- mustalipeästä saostettua ligniiniä. Diplomityön ensimmäisenä tavoitteena oli löytää menetelmä, jolla voidaan poistaa kraft ligniinistä tuhkaa. Työssä raaka-aineena käytetyn saostetun ligniinin tuhkapitoisuus oli noin 3 %. Tarkoituksena oli saada laskettua tuhkapitoisuutta uudelleenlieton, suodatuksen ja pesun avulla. Työn toisena tavoitteena oli hiilihydraattien poisto kraft ligniinistä. Hiilihydraatit, pääosin hemiselluloosaa, ovat kiinnittyneet ligniiniin vahvoin kovalenttisin sidoksin. Aiempien kokemusten perusteella hemiselluloosat eivät irtoa ligniinistä vesipesun yhteydessä, vaan niiden irrottaminen vaatii onnistuakseen happo-, entsyymi- tai mikrobikäsittelyn, mikäli halutaan säilyttää ligniinin rakenne muuttumattomana. Tässä työssä käytetyt kraft ligniinin puhdistusmenetelmät olivat lietto, happohydrolyysi ja entsymaattinen hydrolyysi, joista kumpikin sisälsi ligniinin uudelleenlieton, suodatuksen ja muodostuneen kakun pesun vedellä.

Biopolttoaineen rinnakkaispolton kannattavuustarkastelu hiilipölypolttokattilassa Martinlaakso 2:ssa

Euroopan unionin asettamat tavoitteet kasvihuonepäästöjen vähennykselle johtavat vih-reämpään teknologiaan. Tämä diplomityö on teoreettinen tutkimus, joka käsittelee biopolt-toaineen rinnakkaispolton kannattavuutta Vantaan Energian Martinlaakso 2:sen hiilipöly-polttokattilassa. Työssä perehdytään viiteen eri biopolttoainevaihtoehtoon, joita tarkastellaan viidessä eri skenaariossa, jotka vastaavat: 10, 20, 30, 40 ja 50 % biopolttoaineen osuutta kattilassa tuo-tetusta energiasta. Skenaarioissa on pohdittu tarvittavia investointikustannuksia ja muutos-töitä hiilipölypolttokattilassa. Tutkimuksessa on huomioitu myös uusi isojen laitosten pääs-töjä koskeva direktiivi, kattilan oletettava käyttöikä sekä biopolttoaineiden tuet. Saaduista arvioista on lopuksi laskettu vuosittainen polttoainekohtainen kustannusarvio ja investoin-nin kannattavuusarvio. Tuloksista voidaan päätellä, että sahanpurun mahdollisimman suuri hyötykäyttö on kannat-tavaa. Mikäli halutaan käyttää suuria määriä biopolttoainetta, (yli 20 % tuotetusta energias-ta) ei sahanpuru ole varteenotettava vaihtoehto huonon saatavuutensa johdosta. Tällöin hakkeen kaasutuslaitos olisi paras ratkaisu, mutta laitoksen kannattavuus riippuu tulevista energiatuista. Ilman energiatukia sahanpurun hyötykäyttö on ainoa kannattava investointi.

The Dual Role of HIF Hydroxylase PHD3 in Cancer Cell Survival

Most advanced tumours face periods of reduced oxygen availability i.e. hypoxia. During these periods tumour cells undergo adaptive changes enabling their survival under adverse conditions. In cancer hypoxia-induced cellular changes cause tumour progression, hinder cancer treatment and are indicative of poor prognosis. Within cells the main regulator of hypoxic responses is the hypoxia-inducible factor (HIF). HIF governs the expression of over a hundred hypoxia-inducible genes that regulate a number of cellular functions such as angiogenesis, glucose metabolism and cell migration. Therefore the activity of HIF must be tightly governed. HIF is regulated by a family of prolyl hydroxylase enzymes, PHDs, which mark HIF for destruction in normoxia. Under hypoxic conditions PHDs lose much of their enzymatic activity as they need molecular oxygen as a cofactor. Out of the three PHDs (PHD1, 2 and 3) PHD2 has been considered to be the main HIF-1 regulator in normoxic conditions. PHD3 on the other hand shows the most robust induction in response to oxygen deprivation and it has been implied as the main HIF-1 regulator under prolonged hypoxia. SQSTM1/p62 (p62) is an adaptor protein that functions through its binding motifs to bring together proteins in order to regulate signal transduction. In non-stressed situations p62 levels are kept low but its expression has been reported to be upregulated in many cancers. It has a definitive role as an autophagy receptor and as such it serves a key function in cancer cell survival decisions. In my thesis work I evaluated the significance of PHD3 in cancer cell and tumour biology. My results revealed that PHD3 has a dual role in cancer cell fate. First, I demonstrated that PHD3 forms subcellular protein aggregates in oxygenated carcinoma cells and that this aggregation promotes apoptosis induction in a subset of cancer cells. In these aggregates an adaptor protein SQSTM1/p62 interacts with PHD3 and in so doing regulates PHD3 expression. SQSTM1/p62 expression is needed to keep PHD3 levels low in normoxic conditions. Its levels rapidly decrease in response to hypoxia allowing PHD3 protein levels to be upregulated and the protein to be diffusely expressed throughout the cell. The interaction between PHD3 and SQSTM1/p62 limits the ability of PHD3 to function on its hydroxylation target protein HIF-1alpha. Second, the results indicate that when PHD3 is upregulated under hypoxia it protects cancer cells by allowing cell cycle to proceed from G1 to S-phase. My data demonstrates that PHD3 may either cause cell death or protect the cells depending on its expression pattern and the oxygen availability of tumours.

Development of racemization catalysts for dynamic kinetic resolution

Preparation of optically active compounds is of high importance in modern medicinal chemistry. Despite recent advances in the field of asymmetric synthesis, resolution of racemates still remains the most utilized way for preparation of single enantiomers in industrial scale due to its cost-efficiency and simplicity. Enzymatic kinetic resolution (KR) of racemates is a classical method for separation of enantiomers. One of its drawbacks is the limitation of target enantiomer yield to 50%. Dynamic Kinetic Resolution (DKR) allows to reach yields up to 100% by in situ racemization of the less reactive enantiomer. In the first part of this thesis, a number of half-sandwich ruthenium complexes were prepared and evaluated as catalysts for racemization of optically active secondary alcohols. A leading catalyst, Bn5CpRu(CO)2Cl, was identified. The catalyst discovered was extensively characterized by its application for DKR of a broad range of secondary alcohols in a wide range of reaction loadings (1 mmol – 1 mol). Cost-efficient chromatography-free procedure for preparation of this catalyst was developed. Further, detailed kinetic and mechanistic studies of the racemization reactions were performed. Comparison of racemization rates in the presence of Bn5CpRu(CO)2Cl and Ph5CpRu(CO)2Cl catalysts reveals that the performance of the catalytic system can be adjusted by matching of the electronic properties of the catalysts and the substrates. Moreover, dependence of the rate-limiting step from the electronic properties of the reagents was observed. Important conclusions about reaction mechanism were made. Finally, an alternative approach to DKR of amines based on space separated vessels was addressed. This procedure allows the combination of thermolabile enzyme with racemization catalysts active only at high temperatures.

Studies on chemoenzymatic synthesis: Lipase-catalyzed acylation in multistep organic synthesis

In this thesis, biocatalysis is defined as the science of using enzymes as catalysts in organic synthesis. Environmental aspects and the continuously expanding repertoire of available enzymes have firmly established biocatalysis as a prominent means of chemo-, regio- and stereoselective synthesis. Yet, no single methodology can solve all the challenges faced by a synthetic chemist. Therefore, the knowledge and the skills to combine different synthetic methods are relevant. Lipases are highly useful enzymes in organic synthesis. In this thesis, an effort is being made to form a coherent picture of when and how can lipases be incorporated into nonenzymatic synthesis. This is attempted both in the literature review and in the discussion of the results presented in the original publications contained in the thesis. In addition to lipases, oxynitrilases were also used in the work. The experimental part of the thesis comprises of the results reported in four peer-reviewed publications and one manuscript. Selected amines, amino acids and sugar-derived cyanohydrins or their acylated derivatives were each prepared in enantio- or diastereomerically enriched form. Where applicable, attempts were made to combine the enzymatic reactions to other synthetic steps either by the application of completely separate sequential reactions with isolated intermediates (kinetic and functional kinetic resolution of amines), simultaneously occurring reactions without intermediate isolation (dynamic kinetic resolution of amino acid esters) or sequential reactions but without isolating the intermediates (hydrocyanation of sugar aldehydes with subsequent diastereoresolution). In all cases, lipase-catalyzed acylation was the key step by which stereoselectivity was achieved. Lipase from Burkholderia cepacia was a highly selective enzyme with each substrate category, but careful selection of the acyl donor and the solvent was important as well.

Production of Mg(OH)2 from Mg-silicate rock for CO2 mineral sequestration

Sequestration of carbon dioxide in mineral rocks, also known as CO2 Capture and Mineralization (CCM), is considered to have a huge potential in stabilizing anthropogenic CO2 emissions. One of the CCM routes is the ex situ indirect gas/sold carbonation of reactive materials, such as Mg(OH)2, produced from abundantly available Mg-silicate rocks. The gas/solid carbonation method is intensively researched at Åbo Akademi University (ÅAU ), Finland because it is energetically attractive and utilizes the exothermic chemistry of Mg(OH)2 carbonation. In this thesis, a method for producing Mg(OH)2 from Mg-silicate rocks for CCM was investigated, and the process efficiency, energy and environmental impact assessed. The Mg(OH)2 process studied here was first proposed in 2008 in a Master’s Thesis by the author. At that time the process was applied to only one Mg-silicate rock (Finnish serpentinite from the Hitura nickel mine site of Finn Nickel) and the optimum process conversions, energy and environmental performance were not known. Producing Mg(OH)2 from Mg-silicate rocks involves a two-staged process of Mg extraction and Mg(OH)2 precipitation. The first stage extracts Mg and other cations by reacting pulverized serpentinite or olivine rocks with ammonium sulfate (AS) salt at 400 - 550 oC (preferably < 450 oC). In the second stage, ammonia solution reacts with the cations (extracted from the first stage after they are leached in water) to form mainly FeOOH, high purity Mg(OH)2 and aqueous (dissolved) AS. The Mg(OH)2 process described here is closed loop in nature; gaseous ammonia and water vapour are produced from the extraction stage, recovered and used as reagent for the precipitation stage. The AS reagent is thereafter recovered after the precipitation stage. The Mg extraction stage, being the conversion-determining and the most energy-intensive step of the entire CCM process chain, received a prominent attention in this study. The extraction behavior and reactivity of different rocks types (serpentinite and olivine rocks) from different locations worldwide (Australia, Finland, Lithuania, Norway and Portugal) was tested. Also, parametric evaluation was carried out to determine the optimal reaction temperature, time and chemical reagent (AS). Effects of reactor types and configuration, mixing and scale-up possibilities were also studied. The Mg(OH)2 produced can be used to convert CO2 to thermodynamically stable and environmentally benign magnesium carbonate. Therefore, the process energy and life cycle environmental performance of the ÅAU CCM technique that first produces Mg(OH)2 and the carbonates in a pressurized fluidized bed (FB) were assessed. The life cycle energy and environmental assessment approach applied in this thesis is motivated by the fact that the CCM technology should in itself offer a solution to what is both an energy and environmental problem. Results obtained in this study show that different Mg-silicate rocks react differently; olivine rocks being far less reactive than serpentinite rocks. In summary, the reactivity of Mg-silicate rocks is a function of both the chemical and physical properties of rocks. Reaction temperature and time remain important parameters to consider in process design and operation. Heat transfer properties of the reactor determine the temperature at which maximum Mg extraction is obtained. Also, an increase in reaction temperature leads to an increase in the extent of extraction, reaching a maximum yield at different temperatures depending on the reaction time. Process energy requirement for producing Mg(OH)2 from a hypothetical case of an iron-free serpentine rock is 3.62 GJ/t-CO2. This value can increase by 16 - 68% depending on the type of iron compound (FeO, Fe2O3 or Fe3O4) in the mineral. This suggests that the benefit from the potential use of FeOOH as an iron ore feedstock in iron and steelmaking should be determined by considering the energy, cost and emissions associated with the FeOOH by-product. AS recovery through crystallization is the second most energy intensive unit operation after the extraction reaction. However, the choice of mechanical vapor recompression (MVR) over the “simple evaporation” crystallization method has a potential energy savings of 15.2 GJ/t-CO2 (84 % savings). Integrating the Mg(OH)2 production method and the gas/solid carbonation process could provide up to an 25% energy offset to the CCM process energy requirements. Life cycle inventory assessment (LCIA) results show that for every ton of CO2 mineralized, the ÅAU CCM process avoids 430 - 480 kg CO2. The Mg(OH)2 process studied in this thesis has many promising features. Even at the current high energy and environmental burden, producing Mg(OH)2 from Mg-silicates can play a significant role in advancing CCM processes. However, dedicated future research and development (R&D) have potential to significantly improve the Mg(OH)2 process performance.

Biosynthesis of pyranonaphthoquinone polyketides:characterization of the alnumycin pathway

Alnumycin A is an aromatic pyranonaphthoquinone (PNQ) polyketide closely related to the model compound actinorhodin. While some PNQ polyketides are glycosylated, alnumycin A contains a unique sugar-like dioxane moiety. This unusual structural feature made alnumycin A an interesting research target, since no information was available about its biosynthesis. Thus, the main objective of the thesis work became to identify the steps and the enzymes responsible for the biosynthesis of the dioxane moiety. Cloning, sequencing and heterologous expression of the complete alnumycin gene cluster from Streptomyces sp. CM020 enabled the inactivation of several alnumycin biosynthetic genes and preliminary identification of the gene products responsible for pyran ring formation, quinone formation and dioxane biosynthesis. The individual deletions of the genes resulted in the production of several novel metabolites, which in many cases turned out to be pathway intermediates and could be used for stepwise enzymatic reconstruction of the complete dioxane biosynthetic pathway in vitro. Furthermore, the in vitro reactions with purified alnumycin biosynthetic enzymes resulted in the production of other novel compounds, both pathway intermediates and side products. Identification and molecular level studies of the enzymes AlnA and AlnB catalyzing the first step of dioxane biosynthesis – an unusual C-ribosylation step – led to a mechanistic proposal for the C-ribosylation of the polyketide aglycone. The next step on the dioxane biosynthetic pathway was found to be the oxidative conversion of the attached ribose into a highly unusual dioxolane unit by Aln6 belonging to an uncharacterized protein family, which unexpectedly occurred without any apparent cofactors. Finally, the last step of the pathway was found to be catalyzed by the NADPH-dependent reductase Aln4, which is able to catalyze the conversion of the formed dioxolane into a dioxane moiety. The work presented here and the knowledge gained of the enzymes involved in dioxane biosynthesis enables their use in the rational design of novel compounds containing C–C bound ribose, dioxolane and dioxane moieties.

Puuperäisten biomassahydrolysaattien likaamien membraanien pesu

Tässä diplomityössä tutkittiin biomassahydrolysaatilla liattujen ultrasuodatusmembraanien pesua. Työn kirjallisuusosassa käsitellään membraanien likaantumista ja pesua, puuperäistä biomassahydrolysaattia sekä biomassahydrolysaatin suodatusta membraaneilla ja membraanien karakterisointia. Kokeellisessa osassa tutkittiin pesuaineiden vaikutusta koivuhydrolysaatilla liattujen membraanien peseytyvyyteen. Käytetyt ultrasuodatusmembraanit olivat Alfa Lavalin UFX5 ja ETNA01PP. Membraanien pesussa käytettiin Ecolabin entsymaattisia P3-ultrasil 53 ja P3-ultrasil 67 pesuaineita yhdessä emäksisen P3-ultrasil 69 pesuaineen kanssa sekä hapanta P3-ultrasil 75 pesuainetta. Lisäksi emäksisiä P3-ultrasil 110, P3-ultrasil 112 ja P3-ultrasil 115 pesuaineita. Emäksisten pesujen tehokkuutta vertailtiin pelkällä natriumhydroksidilla tehtyyn pesuun. Membraanien likaantumista arvioitiin mittaamalla puhtaan veden vuota ennen ja jälkeen likaantumissuodatuksen ja laskemalla niiden vuoarvojen erotus. Samalla tavalla arvioitiin pesuaineiden vaikutusta membraanin peseytyvyyteen. Lisäksi peseytyvyyttä arvioitiin FTIR–analyyseillä, kontaktikulmamittauksilla ja membraanin sisältämän ligniinin värjäyksellä. Tutkimuksissa havaittiin, että koivuhydrolysaatilla liatuille UFX5 membraaneille vesivuon sekä FTIR–spektrin muuttumisen perusteella sopii parhaiten emäksisen ja happaman pesuaineen kaksivaiheinen pesu. Kontaktikulman palautumisen perusteella parhaiten sopi pelkällä emäksisellä P3-Ultrasil 115 tehty pesu. Ligniiniä parhaiten, mikroskooppiin liitetyllä kameralla otettujen kuvien perusteella, poisti entsymaattinen P3-ultrasil 67, mutta tämä samalla modifioi membraanin pintaa. Tässä tutkimuksessa havaittiin, että emäksinen ja entsymaattinen pesuaine pesevät melko hyvin koivuhydrolysaatilla liattuja membraaneja. Lisäksi havaittiin, että ligniini tunkeutuu suodatuksissa membraanin sisään, joten pelkkä pesuaineen kierrättäminen laitteistossa ei riitä vaan tarvitaan paineellista pesua.

Effects of cytochrome P450 enzyme inhibitors and inducers on the metabolism of S-ketamine

The human body eliminates foreign compounds primarily by metabolizing them to hydrophilic forms to facilitate effective excretion through the kidneys. Cytochrome P450 (CYP) enzymes in the liver and intestine contribute to the metabolism of many drugs. Pharmacokinetic drugdrug interactions occur if the activity of CYPs are inhibited or induced by another drug. Prescribing multiple drugs to the improve effectiveness of therapy or to treat coexisting diseases is a common practice in clinical medicine. Polypharmacy predisposes patients to adverse effects because of the profound unpredictability in CYP enzymatic-mediated drug metabolism. S-ketamine is a phencyclidine derivative which functions as an antagonist of the N-methyl-Daspartate (NMDA) receptor in the central nervous system. It is a unique anaesthetic producing “dissociative anaesthesia” in high doses and analgesia in low doses. Studies with human liver microsomes suggest that ketamine is metabolized primarily via CYP3A4 and CYP2B6 enzymes. In this thesis, in healthy volunteers, randomized and controlled cross-over studies were conducted to investigate the effects of different CYP inducers and inhibitors on the pharmacokinetics and pharmacodynamics of oral and intravenous S-ketamine. The plasma concentrations of ketamine and its metabolite, norketamine, were determined at different timepoints over a 24 hour period. Other pharmacodynamic variables were examined for 12 hours. Results of these studies showed that the inhibition of the CYP3A4 pathway by clarithromycin or grapefruit juice increased the exposure to oral S-ketamine by 2.6- and 3.0-fold. Unexpectedly, CYP3A4 inhibition by itraconazole caused no significant alterations in the plasma concentrations of oral S-ketamine. CYP3A4 induction by St. John´s wort or rifampicin decreased profoundly the concentrations of oral S-ketamine. However, after rifampicin, there were no significant differences in the plasma concentrations of S-ketamine when it was administered intravenously. This demonstrated that rifampicin inhibited the metabolism of Sketamine at the intestinal level. When CYP2B6 was inhibited by ticlopidine, there was a 2.4- fold increase in the exposure of S-ketamine. These studies demonstrated that low dose oral Sketamine is metabolized both via CYP3A4 and CYP2B6 pathways. The concomitant use of drugs that affect CYP3A4 or CYP2B6, during oral S-ketamine treatment, may cause clinically significant drug-drug interactions.

Regioselective modification of galactose-containing polysaccharides in aqueous media

Biorefining is defined as sustainable conversion of biomass into marketable products and energy. Forests cover almost one third of earth’s land area, and account for approximately 40% of the total annual biomass production. In forest biorefining, the wood components are, in addition to the traditional paper and board products, converted into chemicals and biofuels. The major components in wood are cellulose, hemicelluloses, and lignin. The main hemicellulose in softwoods, which are of interest especially for the Nordic forest industry, is O-acetyl galactoglucomannan (GGM). GGM can be isolated in industrial scale from the waste waters of the mechanical pulping process, but is not yet today industrially utilized. In order to attain desired properties of GGM for specific end-uses, chemical and enzymatic modifications can be performed. Regioselective modifications of GGM, and other galactose-containing polysaccharides were done by oxidations, and by combining oxidations with subsequent derivatizations of the formed carbonyl or carboxyl groups. Two different pathways were investigated: activation of the C-6 positions in different sugar units by TEMPO-mediated oxidation, and activation of C-6 position in only galactose-units by oxidation catalyzed by the enzyme galactose oxidase. The activated sites were further selectively derivatized; TEMPO-oxidized GGM by a carbodiimide-mediated reaction forming amides, and GO-oxidized GGM by indium-mediated allylation introducing double or triple bonds to the molecule. In order to better understand the reaction, and to develop a MALDI-TOF-MS method for characterization of regioselectively allylated GGM, α-D-galactopyranoside and raffinose were used as model compounds. All reactions were done in aqueous media. To investigate the applicability of the modified polysaccharides for, e.g., cellulose surface functionalization, their sorption onto pulp fibres was studied. Carboxylation affects the sorption tendency significantly; a higher degree of oxidation leads to lower sorption. By controlling the degree of oxidation of the polysaccharides and the ionic strength of the sorption media, high degrees of sorption of carboxylated polysaccharides onto cellulose could, however, be obtained. Anionic polysaccharides were used as templates during laccase-catalyzed polymerization of aniline, offering a green, chemo-enzymatic route for synthesis of conducting polyaniline (PANI) composite materials. Different polysaccharide templates, such as, native GGM, TEMPO-oxidized GGM, naturally anionic κ-carrageenan, and nanofibrillated cellulose produced by TEMPO-oxidation, were assessed. The conductivity of the synthesized polysaccharide/PANI biocomposites varies depending on the polysaccharide template; κ-CGN, the anionic polysaccharide with the lowest pKa value, produces the polysaccharide/PANI biocomposites with the highest conductivity. The presented derivatization, sorption, and polymerization procedures open new application windows for polysaccharides, such as spruce GGM. The modified polysaccharides and the conducting biocomposites produced provide potential applications in biosensors, electronic devices, and tissue engineering.