Immobilization of Burkholderia cepacia Lipase: Kinetic Resolution in Organic Solvents, Ionic Liquids and in Their Mixtures

Immobilization of Burkholderia cepacia Lipase: Kinetic Resolution in Organic Solvents, Ionic Liquids and in Their Mixtures Biocatalysis opens the door to green and sustainable processes in synthetic chemistry allowing the preparation of single enantiomers, since the enzymes are chiral and accordingly able to catalyze chemical reactions under mild conditions. Immobilization of enzymes enhances process robustness, often stabilizes and activates the enzyme, and enables reuse of the same enzyme preparation in multiple cycles. Although hundreds of variations of immobilization methods exist, there is no universal method to yield the highly active, selective and stable enzyme catalysts. Therefore, new methods need to be developed to obtain suitable catalysts for different substrates and reaction environments. Lipases are the most widely used enzymes in synthetic organic chemistry. The literature part together with the experimental part of this thesis discusses of the effects of immobilization methods mostly used to enhance lipase activity, stability and enantioselectivity. Moreover, the use of lipases in the kinetic resolution of secondary alcohols in organic solvents and in ionic liquids is discussed. The experimental work consists of the studies of immobilization of Burkholderia cepacia lipase (lipase PS) using three different methods: encapsulation in sol-gels, cross-linked enzyme aggregates (CLEAs) and supported ionic liquids enzyme catalysts (SILEs). In addition, adsorption of lipase PS on celite was studied to compare the results obtained with sol-gels, CLEAs and SILEs. The effects of immobilization on enzyme activity, enantioselectivity and hydrolysis side reactions were studied in kinetic resolution of three secondary alcohols in organic solvents, in ionic liquids (ILs), and in their mixtures. Lipase PS sol-gels were shown to be active and stable catalysts in organic solvents and solvent:IL mixtures. CLEAs and SILEs were highly active and enantioselective in organic solvents. Sol-gels and SILEs were reusable in several cycles. Hydrolysis side reaction was suppressed in the presence of sol-gels and CLEAs.

Bio and chemocatalysis for stereo and regioselective one-pot reaction applications

The development of cost efficient, selective and sustainable chemical processes for production of chiral building blocks is of great importance in synthetic and industrial organic chemistry. One way to reach these objectives is to carry out several reactions steps in one vessel at one time. Furthermore, when this kind of one-pot multi step reactions are catalyzed by heterogeneous chemo- and bio-catalysts, which can be separated from the reaction products by filtration, practical access to chiral small molecules for further utilization can be obtained. The initial reactions studied in this thesis are the two step dynamic kinetic resolution of rac-2-hydroxy-1-indanone and the regioselective hydrogenation of 1,2-indanedione. These reactions are then combined in a new heterogeneously catalyzed one-pot reaction sequence enabling simple recovery of the catalysts by filtration, facilitating simple reaction product isolation. Conclusively, the readily available 1,2-indanedione is by the presented one-pot sequence, utilizing heterogeneous enzyme and transition metal based catalysts, transferred with high regio- and stereoselectivity to a useful chiral vicinal hydroxyl ketone structure. Additional and complementary investigation of homogeneous half-sandwich ruthenium complexes for catalyzing the epimerization of chiral secondary alcohols of five natural products containing additional non-functionalized stereocenters was conducted. In principle, this kind of epimerization reactions of single stereocenters could be utilized for converting inexpensive starting materials, containing other stereogenic centers, into diastereomeric mixtures from which more valuable compounds can be isolated by traditional isolation techniques.

Determination of sugars and organic acids in pulp samples by capillary electrophoresis with UV detection

This MSc work was done in the project of BIOMECON financed by Tekes. The prime target of the research was, to develop methods for separation and determination of carbohydrates (sugars), sugar acids and alcohols, and some other organic acids in hydrolyzed pulp samples by capillary electrophoresis (CE) using UV detection. Aspen, spruce, and birch pulps are commonly used for production of papers in Finland. Feedstock components in pulp predominantly consist of carbohydrates, organic acids, lignin, extractives, and proteins. Here in this study, pulps have been hydrolyzed in analytical chemistry laboratories of UPM Company and Lappeenranta University in order to convert them into sugars, acids, alcohols, and organic acids. Foremost objective of this study was to quantify and identify the main and by-products in the pulp samples. For the method development and optimization, increased precision in capillary electrophoresis was accomplished by calculating calibration data of 16 analytes such as D-(-)-fructose, D(+)-xylose, D(+)-mannose, D(+)-cellobiose, D-(+)-glucose, D-(+)-raffinose, D(-)-mannitol, sorbitol, rhamnose, sucrose, xylitol, galactose, maltose, arabinose, ribose, and, α-lactose monohydratesugars and 16 organic acids such as D-glucuronic, oxalic, acetic, propionic, formic, glycolic, malonic, maleic, citric, L-glutamic, tartaric, succinic, adipic, ascorbic, galacturonic, and glyoxylic acid. In carbohydrate and polyalcohol analyses, the experiments with CE coupled to direct UV detection and positive separation polarity was performed in 36 mM disodium hydrogen phosphate electrolyte solution. For acid analyses, CE coupled indirect UV detection, using negative polarity, and electrolyte solution made of 2,3 pyridinedicarboxylic acid, Ca2+ salt, Mg2+ salts, and myristyltrimethylammonium hydroxide in water was used. Under optimized conditions, limits of detection, relative standard deviations and correlation coefficients of each compound were measured. The optimized conditions were used for the identification and quantification of carbohydrates and acids produced by hydrolyses of pulp. The concentrations of the analytes varied between 1 mg – 0.138 g in liter hydrolysate.

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).

First dice your dill – new methods and techniques in sample handling

This book is dedicated to celebrate the 60th birthday of Professor Rainer Huopalahti. Professor Rainer “Repe” Huopalahti has had, and in fact is still enjoying a distinguished career in the analysis of food and food related flavor compounds. One will find it hard to make any progress in this particular field without a valid and innovative sample handling technique and this is a field in which Professor Huopalahti has made great contributions. The title and the front cover of this book honors Professor Huopahti’s early steps in science. His PhD thesis which was published on 1985 is entitled “Composition and content of aroma compounds in the dill herb, Anethum graveolens L., affected by different factors”. At that time, the thesis introduced new technology being applied to sample handling and analysis of flavoring compounds of dill. Sample handling is an essential task that in just about every analysis. If one is working with minor compounds in a sample or trying to detect trace levels of the analytes, one of the aims of sample handling may be to increase the sensitivity of the analytical method. On the other hand, if one is working with a challenging matrix such as the kind found in biological samples, one of the aims is to increase the selectivity. However, quite often the aim is to increase both the selectivity and the sensitivity. This book provides good and representative examples about the necessity of valid sample handling and the role of the sample handling in the analytical method. The contributors of the book are leading Finnish scientists on the field of organic instrumental analytical chemistry. Some of them are also Repe’ s personal friends and former students from the University of Turku, Department of Biochemistry and Food Chemistry. Importantly, the authors all know Repe in one way or another and are well aware of his achievements on the field of analytical chemistry. The editorial team had a great time during the planning phase and during the “hard work editorial phase” of the book. For example, we came up with many ideas on how to publish the book. After many long discussions, we decided to have a limited edition as an “old school hard cover book” – and to acknowledge more modern ways of disseminating knowledge by publishing an internet version of the book on the webpages of the University of Turku. Downloading the book from the webpage for personal use is free of charge. We believe and hope that the book will be read with great interest by scientists working in the fascinating field of organic instrumental analytical chemistry. We decided to publish our book in English for two main reasons. First, we believe that in the near future, more and more teaching in Finnish Universities will be delivered in English. To facilitate this process and encourage students to develop good language skills, it was decided to be published the book in English. Secondly, we believe that the book will also interest scientists outside Finland – particularly in the other member states of the European Union. The editorial team thanks all the authors for their willingness to contribute to this book – and to adhere to the very strict schedule. We also want to thank the various individuals and enterprises who financially supported the book project. Without that support, it would not have been possible to publish the hardcover book.

Fractionation of Hen Egg and Oat Lipids with supercritical Fluids Chemical and functional Properties of Fractions

Hen eggs and oats (Avena Sativa) are important materials for the food industry. Today, instead of merely satisfying the feeling of hunger, consumers are asking for healthier, biologically active and environmentally friendly products. The growing awareness of consumers’ increasing demands presents a great challenge to the food industry to develop more sustainable products and utilise modern and effective techniques. The modification of yolk fatty acid composition by means of feed supplements is well understood. Egg yolk phospholipids are polar lipids and are used in several applications including food, cosmetics, pharmaceuticals, and special nutrients. Egg yolk phospholipids are excellent emulsifiers, typically sold as mixtures of phospholipids, triacylglycerols, and cholesterol. However, highly purified and characterised phospholipids are needed in several sophisticated applications. Industrial fractionation of phospholipids is usually based on organic solvents. With these fractionation techniques, some harmful residues of organic solvents may cause problems in further processing. The objective of the present study was to investigate the methods to improve the functional properties of eggs, to develop techniques to isolate the fractions responsible for the specific functional properties of egg yolk lipids, and to apply the developed techniques to plant-based materials, too. Fractionation techniques based on supercritical fluids were utilised for the separation of the lipid fractions of eggs and oats. The chemical and functional characterisation of the fractions were performed, and the produced oat polar lipid fractions were tested as protective barrier in encapsulation processes. Modifying the fatty acid compositions of egg yolks with different types of oil supplements in feed had no affect on their functional or sensory properties. Based on the results of functional and sensory analysis, it is evident that eggs with modified fatty acid compositions are usable in several industrial applications. These applications include liquid egg yolk products used in mayonnaise and salad dressings. Egg yolk powders were utilised in different kinds of fractionation processes. The precipitation method developed in this study resembles the supercritical anti-solvent method, which is typically used in the pharmaceutical industry. With pilot scale supercritical fluid processes, non-polar lipids and polar lipids were successfully separated from commercially produced egg yolk powder and oat flakes. The egg and oat-based polar lipid fractions showed high purities, and the corresponding delipidated fractions produced using supercritical techniques offer interesting starting materials for the further production of bioactive compounds. The oat polar lipid fraction contained especially digalactosyadiacylglycerol, which was shown to have valuable functional properties in the encapsulation of probiotics.

Pulsed corona discharge as an advanced oxidation process for degradation of organic

Advanced oxidation processes (AOPs) have been studied and developed to suffice the effective removal of refractory and toxic compounds in polluted water. The quality and cost of wastewater treatment need improvements, and electric discharge technology has a potential to make a significant difference compared to other established AOPs based on energy efficiency. The generation of active oxidant species such as ozone and hydroxyl radicals by high voltage discharge is a relatively new technology for water treatment. Gas-phase pulsed corona discharge (PCD), where a treated aqueous solution is dispersed between corona-producing electrodes free of the dielectric barriers, was developed as an alternative approach to the problem. The short living radicals and ozone formed in the gas phase and at the gas-liquid interface react with dissolved impurities. PCD equipment has a relatively simple configuration, and with the reactor in an enclosed compartment, it is insensitive towards gas humidity and does not need the gas transport. In this thesis, PCD was used to study and evaluate the energy efficiency for degrading various organic compounds, as well as the chemistry of the oxidation products formed. The experiments investigate the aqueous oxidation of phenol, humic substances, pharmaceutical compounds (paracetamol, ibuprofen, indomethacin, salicylic acids, -estradiol), as well as lignin degradation and transformation to aldehydes. The study aims to establish the influence of initial concentration of the target pollutant, the pulsed discharge parameters, gas phase composition and the pH on the oxidation kinetics and the efficiency. Analytical methods to measure the concentrations of the target compounds and their by-products include HPLC, spectrophotometry, TOC and capillary electrophoresis. The results of the research included in this summary are presented in the attached publications and manuscripts accepted for publication. Pulsed corona discharge proved to be highly effective in oxidizing each of the target compounds, surpassing the closest competitor, conventional ozonation. The increase in oxidation efficiencies for some compounds in oxygen media and at lower pulse repetition frequencies shows a significant role of ozone. The role of the ·OH radicals was established in the surface reactions. The main oxidation products, formation of nitrates, and the lignin transformation were quantified. A compound specific approach is suggested for optimization of the PCD parameters that have the most significant impact on the oxidation energy efficiency because of the different characteristics and responses of the target compound to the oxidants, as well as different admixtures that are present in the wastewater. Further studies in the method’s safety (nitration and nitrosation of organic compounds, nitrite and nitrate formation enhancement) are needed for promoting the method.