As nontoxic biobased surfactants derived from plant oils and cellulose or starch, saccharide-fatty acid esters are widely used in cosmetics, food, and pharmaceutical industries due to their biocompatibility, biodegradability as well as antimicrobial activity. Generally, saccharide-fatty acid esters are synthesized chemically under high pressure, temperature and the presence of alkaline or acid catalysts leading to low-quality products (chemo-degradation of double bonds and oxygenated moieties) and large amounts of byproducts. In contrast, biocatalytic synthesis enhances sustainability: near-ambient pressure and temperature, the absence of toxic, acids and bases catalysts, and improved selectivity of products. For lipase-catalyzed synthesis under nearly anhydrous conditions, the major hurdle to be overcome is the poor miscibility of the acyl donor and acceptor substrates, resulting in slow reaction rates. Although several approaches such as, the employments of organic solvents, complexation agents, and ionic liquids, have been reported in the literature, a robust solution is desperately needed. This study focused on employing immobilized lipases under completely solvent-free conditions to synthesize saccharide-fatty acid esters using the ester products to enhance miscibility. Experimentally, metastable saccharide particles with a diameter of 10-100 micron-sized suspensions of saccharide were formed in oleic acid-rich ester mixtures initially for synthesis of saccharide-fatty acid esters in packed bed bioreactor containing immobilized lipases. Water, a by-product that limits ester yield by promoting hydrolysis, was removed via free evaporation. In this dissertation, a bioreactor system was developed for the eco-friendly solvent-free, immobilized lipase-catalyzed synthesis of biobasaed surfactants utilizing suspensions as reaction medium with 88 wt% in 6 days; the performance of the bioreactor systems developed for Objective 1 was optimized through water concentration control and interval time with 91 wt% in 4.8 days; and to improve design of bioreactor system developed in Objective 1 by in-line filter and derive a mathematical model to describe the production of esters by the bioreactor systems developed. Finally, 84 wt% ester content was achieved in 8.4 days.

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

Biocatalysts offer advantages over their chemical counterparts in terms of their high enantioselectivity and the opportunity to develop more environmentally friendly processes. However, the widespread adoption of biocatalytic processes is hampered by the long development times for enzymes with novel and sufficient activity and adequate stability under operating conditions. Protein engineering, while extremely useful for modifying the properties of protein catalysts in select cases, still cannot be performed rapidly enough for many applications. In order for biocatalysts to become a competitive alternative to chemical catalysts, new tools to make the tailoring of biocatalysts by protein engineering methods speedier and more efficient are necessary. The aim of this work was to develop methods to aid in the faster production of novel biocatalysts. Protein engineering involves two steps: the generation of diversity and the screening or selection of variants with the desired properties. Both of these must be targeted to create a faster protein engineering process. In the case of the former, this work sought to clone and overexpress some template enzymes which would create smaller, more manageable libraries of mutants with a higher likelihood of function by the manipulation of a few focused amino acid residues. For the latter, this work developed and validated a Monte-Carlo simulation model of pooling to increase screening throughput and created a set of vectors to aid in high-throughput screening by eliminating unwanted mutants from the assay procedure entirely.

Chiral a -hydroxy ketones are important structural units in many natural products, biologically active compounds and the hydroxyl group has frequently been used as a reagent directing group, such as for the selective elaboration of aldol products. In this work, enzymatic synthesis of both enantiomers of the a -hydroxy ketones (2-hydroxy indanone, 2-hydroxy tetralone) using Aspergillus niger by selective &amp / #945 / -oxidation of ketones (1-indanone, 1-tetralone) was studied. The &amp / #945 / -oxidation of ketones was carried out by using whole cells of Aspergillus niger in different growth media. A. niger whole cell catalyzed reactions afforded (S)-configurated 2- hydroxy-1-tetralone with %87 e.e. in DMSO at pH 5.0. In addition to this,while (S)-configurated 2-hydroxy-1-indanone with %33 e.e. in pH 8.0 (in DMSO) was synthesized, (R)-configurated-2-hyroxy-1-indanone with %32 e.e. in pH 7.0 ( in DMSO) was synthesized.

The objective of the study presented here was to develop either enzymatic or whole cell mediated green procedures for the syntheses of a-hydroxy ketones. Production of optically active synthons is crucial for the preparation of fine chemicals. Enzymes and whole-cell biocatalysts have proven to be excellent vehicles with their chiral nature for the biotransformations. Under the light of this discussion, firstly benzaldehyde lyase [BAL, (EC 4.1.2.38)] was used in novel C-C bond formation reactions to obtain interesting and biologically important precursors / 2-Hydroxy-1-arylethan-1-ones and functionalized aliphatic acyloin derivatives. All the compounds were obtained with high yields and in the case of aliphatic acyloin derivatives with high enantiomeric excesses (ee&rsquo / s). Another strategy was to use whole cell biocatalysis. A.flavus 200120 was found to be a promising biocatalyst with the ability to catalyze a broad range of reactions / reduction, hydrolysis and deracemization, while another fungus / A. oryzae 5048 was utilized in bioreduction reactions of benzil and its derivatives. Each reaction was investigated, optimized and thus enhanced via medium design. Products were obtained with high yields and ee&rsquo / s. To sum up, in this study novel efficient green procedures were developed to synthesize various ahydroxy ketones with high yield and stereoselectivity. These newly established methods present promising alternatives to classical chemical methodologies.

Modular polyketide synthases (PKSs) are the largest enzymes known to man and are responsible for synthesizing some of the most important human medicines. Their ability to construct stereochemically-rich carbon chains containing diverse substituents has inspired the biosynthetic community to engineer these factories for the in vitro synthesis of a small library of polyketide compounds. New complex polyketides are discovered every year, yet the lack of compound prohibits characterization and testing of these new compounds for medicinal properties. Smaller polyketide compounds generated in vitro could be organically manipulated to generate larger, more complex polyketide natural products and natural product analogs. Chemoenzymatic approaches like this would be extremely beneficial to the scientific community; however, there are still obstacles that must be overcome before the use of PKS for the preparative synthesis of an in vitro generated polyketide library would prove fruitful: purchasing substrates such as methylmalonyl-CoA is cost-prohibitive, PKSs are often difficult to express and purify, and the products generated are typically nonchromophoric. The use of a malonyl-CoA ligase from Streptomyces coelicolor (MatB) was investigated for the enzymatic synthesis of polyketide extender units such as methylmalonyl-CoA (Chapter 2). MatB synthesized a total of 5 CoA-linked extender units in vitro: malonyl-, methylmalonyl-, ethylmalonyl-, hydroxymalonyl- and methoxymalonyl-CoA. Two ternary complex structures of MatB with bound product and leaving group were also solved to sub-2Å resolution. MatB generated extender units were employed in the module-catalyzed synthesis of a triketide pyrone. The selectivity of a PKS module to incorporate a variety of side chains into triketide pyrones was also investigated (Chapter 3). A total of 10 triketide pyrone compounds were synthesized, 5 produced via modular "stuttering" and one possessing a terminal alkyne chemical handle. Lastly, nonchromphoric polyketide products were made visible upon copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) with fluorescent sulforhodamine B azide revealing insights into in vitro reactivites of a PKS module (Chapter 4). The work described in this dissertation has helped advance the scientific community towards procuring an in vitro synthesized polyketide library for future synthetic applications. / text

In recent years, biocatalytical steps in chemical synthesis are becoming increasingly important for economical and environmental-friendly production. In order to evaluate the use of enzymes in a process at Cambrex Karlskoga AB, an expression system was developed for a dehydrogenase. A synthetic gene was cloned into Escherichia coli DH5a cells, using the pTZ19R expression vector, as previously described in the literature. Protein expression was carried out at 25°C, 30°C and 37°C and results were measured using SDS-PAGE and activity assays. To improve expression, the gene was modified in three ways using PCR, yielding eight clones: It was inserted into the pSE420 expression vector, shortened to avoid inclusion body formation and a missing nucleotide was inserted into the sequence. A protocol for inclusion body screening was also developed. Finally, an assay for determining the kinetic constants of dehydrogenase was designed. It is concluded that further experiments must be done to obtain expression of the dehydrogenase and recommendations for additional work are given. / Biokatalytiska processteg har de senaste åren blivit ett allt viktigare inslag i kemisk syntes för att åstadkomma ekonomisk och miljövänlig produktion. För att utvärdera användandet av enzymer i en process hos Cambrex Karlskoga AB utvecklades ett expressionssystem för ett dehydrogenas. En syntetisk gen klonades in i Escherichia coli DH5a och uttrycktes med hjälp av expressionsvektorn pTZ19R, som tidigare finns beskrivet i litteraturen. Proteinuttrycket utfördes vid 25°C, 30°C och 37°C och resultatet mättes med hjälp av SDS-PAGE och aktivitetsmätningar. Genen för dehydrogenaset modifierades på tre sätt, vilket gav upphov till åtta varianter. Genen fördes över till expressionsvektorn pSE420, kortades för att undvika bildning av inklusionskroppar och en nukleotid som fattades från gensekvensen återinfördes. Ett protokoll utarbetades även för undersökning av inklusionskroppar. Till sist sammanställdes en metod för att undersöka de kinetiska konstanterna hos dehydrogenaset. Slutsatsen av arbetet är att fortsatta studier måste utföras för att erhålla uttryck av dehydrogenaset och rekommendationer ges för framtida undersökningar.

Functionalized polymeric membrane based techniques are becoming increasingly popular in biotechnology, food and pharmaceutical industries due to their versatility and hydrodynamic benefits over traditional materials and methods. This research work has been directed towards the development of functionalized polymeric membranes, extensive experimental and theoretical analyses of some of the fundamental aspects of accessibility, membrane fouling and enzyme catalysis, and applications in affinity based bioseparation and biocatalysis. In this research work, the impact of different types of functionalization techniques, such as functionalization of different membrane materials, covalent and electrostatic immobilization, on interaction of various biomolecules and active sites in membrane has been studied in detail. Avidin was used as model biomolecule, and covalently immobilized within acyl anhydride derivatized nylon based membrane. Quantification of the accessibility of covalently immobilized avidin sites was carried out by model biotinylated probe molecules, such as biotin 4-amidobenzoic acid and biotinylated-BSA. This study has been further extended to separate and purify a target protein, HIV-Tat, from a complex mixture of proteins (97-99 % unwanted protein) using avidin-biotin affinity interaction. It has been demonstrated that covalent immobilization of avidin in membranes reduces the accessibility of active sites for probe molecules. Accessibility decreases further for the biotinylated target protein present in the mixture of other unwanted proteins. Affinity based membrane separation of proteins is also associated with decrease in permeate flux due to fouling in membrane structure. Fouling in the membrane has been discussed by analyzing the characteristics of adsorbed protein layer in membrane. In order to improve the accessibility and fouling behavior of affinity separation of Tat protein, a pre-filtration step has been introduced prior to affinity separation. Significant enhancement in accessibility and reduction in fouling has been observed for pre-filtered cases as it removes unwanted proteins prior to affinity interaction. Contribution of the pre-filtration step in reduction of fouling has been elucidated by simple model equations. Improvement in accessibility and fouling behavior reflects in higher separation efficiency (protein recovery) and lower processing time for the pre-filtered cases. Quality of membrane purified Tat protein was examined by different analytical techniques, such as SDS-PAGE, Western Blot and biotin analysis, and then compared with that purified by traditional packed-bead column chromatography. It has been demonstrated that membrane based technique was able to isolate superior quality of pure monomeric Tat protein compare to column chromatographic technique. The other study carried out as a part of this dissertation, has involved development of high capacity, highly active, stable and reusable functionalized membrane domains for electrostatic immobilization of enzymes. Glucose oxidase (GOX) was used as a model enzyme to study the oxidation of glucose to gluconic acid and hydrogen peroxide under convective flow condition. Two different approaches of functionalization of membranes have been presented. In the first approach, alternative electrostatic attachment of cationic and anionic polyelectrolytes was carried out using Layer-By-Layer (LBL) assembly technique within a functionalized nylon based membrane. In the second one, a hydrophobic PVDF membrane was functionalized by in-situ polymerization of acrylic acid. Kinetics of glucose oxidation, effect of pH and flow rate on the activity of GOX was discussed. A comparative study was presented between the activity of free GOX, electrostatically immobilized GOX and covalently immobilized GOX, along with the advantage of convective mode of operation over soaking mode. A novel study has also been conducted on detachment and reattachment of GOX in the same membrane matrix. Further study has been directed towards implementation of the above mentioned immobilized enzymatic system for oxidative dechlorination of chloro-organics. A first time attempt was made to use a 2-stack functionalized membranes system for simultaneous enzymatic production of hydrogen peroxide in first membrane, and oxidative dechlorination of 2, 4, 6-trichlorophenol (TCP) in the Fe+2 immobilized (by ion exchange) second membrane by Fenton reaction. The technique was efficient in destruction of TCP as evident from the overall dechlorination of 70-80 %. This technique provides additional benefit of reusing the same membrane matrices by reattaching fresh GOX and Fe+2.

NRs are enzymes that catalyze the reduction of nitroaromatics to their corresponding nitroso, hydroxylamine, and, in limited cases, amine They have gathered interest in many scientific communities, and are currently actively researched bioremediation and prodrug activation. Here we attempt to utilize them for the purpose of synthesizing substituted aromatic amines that are found in a number of active pharmaceutical ingredients (APIs). As NRs described in the literature have varying product distribution ranges (from those that produce hydroxylamine to others that yield amine) several similar and different NRs were studied for their selectivity. Additionally, a quantitative structure-activity relationship (QSAR) was determined to characterize the substrate specificity of NRs. To employ the use of flavoenzymes in synthesis, multiple reaction- and protein-engineering approaches were devised. One scheme was to establish an enzymo-chemical synthesis where NRs were paired with reducing agents for a chemical reduction. Another method was to create a monomeric NR through directed evolution from ER scaffolds for future immobilization applications. Protein engineering techniques were also utilized on NADH oxidases which we characterized and developed for nicotinamide cofactor regeneration. As a whole, this dissertation expands our current understanding on NRs and demonstrates the possibility of using several flavoenzymes in the synthesis of organic molecules.

This thesis describes the development and rationalization of processes involved in a new methodology developed in our group, minor enantiomer recycling. The first part of the thesis addresses mechanistic studies of one of the reactions involved in minor enantiomer recycling, dual Lewis acid-Lewis base catalyzed acetylcyanation of aldehydes. The methodology uses a combination of a chiral titanium-salen  complex with a tertiary amine as a catalytic  system  in  the enantioselective  synthesis  of  O-acylated  cyanohydrins from aldehydes and ketonitriles. Mechanistic investigations revealed that the rate-determining step in the reaction changes, depending on the nature of the aldehyde that was used. It was also concluded that cyanohydrin is coordinated to the Lewis acid in the acylation step. The second part of the thesis deals with minor enantiomer recycling, a highly selective one-pot recycling system. In a first step the product is formed as a minor and a major enantiomer by asymmetric catalysis. Recycling of the minor enantiomer, by selective kinetic resolution, regenerates the starting material. Continuous addition of a second reagent, also involved in a coupled exergonic process, leads to an increase of both yield and enantiomeric excess. Recycling procedures for the synthesis of O-acylated and O-formylated cyanohydrins have been developed with high yield and high enantiomeric excess of the products. The study includes development of the systems, comparison to other methodologies in asymmetric catalysis, and attempts to understand the processes involved. / <p>QC 20141202</p>