Tesi sul tema "Biocatalyst"

All fossil fuels contain varying levels of sulfur compounds which are undesirable because they cause environmental pollution, corrosion, acid rain and lead to health problems. There is strict international legislation for the permissible levels of sulfur compounds in fossil fuels. The aim of this research therefore was the biocatalyst development for biodesulfurisation using two approaches. In the first approach, Rhodococcus erythropolis IGTS8-5 and IGTS8-5G were immobilised in porous coke particles and tested in repeated cycles successfully. Both bacterial strains grew well in the chemically defined medium with glucose as the main carbon and energy source and the model sulfur compound dibenzothiophene (DBT) as the sole sulfur source. 0.8 g of cells was immobilized on 250 g of coke particles without refreshing the medium over 72 h while 1.8 g of cells were immobilised on 250 g of coke when the media was refreshed every 24 hours for 120 h after the initial immobilisation batch of 72h. The latter, were used repeatedly in twelve consequtive batch desulfurisation cycles during which the biodesulfurisation activity progressively decreased from over 95% removal of 100 ppm DBT to around 45% removal. DBT removal is often expressed in terms of 2-hydroxybiphenyl which is the end product of biodesulfurisation. The biodesulfurisation activityof immobilised bacteria was equivalent to 310 umol 2-HBP h-1g-1 dry cell weight during the first hour. Freely suspended cells on the other hand exhibited biodesulfurisation activity equivalent to 91 umol 2-HBP h-1g-1 dry cell weight. Unfortunately, after the first 24 h, the activity of the immobilised cells decreased to 12 umol 2-HBP h-1g-1 dry cell weight. Use of plant cell cultures for biodesulfurisation is the other novel aspect of this work. Armoracia rusticana (horse radish) cell culture was chosen as the novel biocatalyst since this plant is a well known source of peroxidase enzyme which is involved in the biodesulfurisation metabolism according to the literature on bacterial biodesulfurisation. Arabidospsis thaliana (thale cress) was also used since its genome is completely sequenced and it is a model organism in genomics studies. Our results indicate that cell suspensions of both plants did show biodesulfurisation activity by reducing the level of sulfur compounds, mainly DBT and other three derivatives from both aqueous and oil phase. When compared to the bacteria, in terms of DBT consumption, the activity of A. rusticana was calculated as 55 umol DBT h-1 g-1 DCW and 65 umol DBT h-1 g-1 DCW for A. thaliana while in bacteria it was 91 umol DBT h-1 g-1 DCW for IGTS8-5 and 73 umol DBT h-1 g-1 DCW for IGTS8-5G. Transcriptomics analysis of the plant cell cultures after exposure to the DBT when compared to control cultures showed alterations in gene expression levels several of which were related to sulfur metabolism and transmembrane transporters of sulfate.

The enzymatic production of R-phenylacetylcarbinol (R-PAC), with either whole cells or partially purified pyruvate decarboxylase (PDC) as the biocatalyst, requires high PDC activity and an inexpensive source of pyruvate for an economical feasible biotransformation process. Microbial pyruvate produced by a vitamin auxotrophic strain of Candida glabrata was selected as a potential substrate for biotransformation. With an optimal thiamine concentration of 60 ??g/l, a pyruvic acid concentration of 43 g/l and yield of 0.42 g/g glucose consumed were obtained. Using microbially-produced unpurified pyruvate resulted in similar PAC concentrations to those with commercial pure substrate confirming its potential for enzymatic PAC production. To obtain high activity yeast PDC, Candida utilis was cultivated in a controlled bioreactor. Optimal conditions for PDC production were identified as: fermentative cell growth at initial pH at 6.0 followed by pH downshift to 3.0. Average specific PDC carboligase activity of 392 ?? 20 U/g DCW was achieved representing a 2.7-fold increase when compared to a constant pH process. A mechanism was proposed in which the cells adapted to the pH decrease by increasing PDC activity to convert the accumulated internal pyruvic acid via acetaldehyde to ethanol thereby reducing intracellular acidification. The effect of pH shift on specific PDC activity of Saccharomyces cerevisiae achieved a comparable increase of specific PDC carboligase activity to 335 U/g DCW. The effect of pyruvic acid at pH 3.0 on induction of PDC activity was confirmed by cultivation at pH 3 with added pyruvic acid. Using microarray techniques, genome-wide transcriptional analyses of the effect of pH shift on S. cerevisiae revealed a transient increased expression of PDC1 after pH shift, which corresponded to the increase in specific PDC activity (although the latter was sustained for a longer period). The results showed significant gene responses to the pH shift with approximately 39 % of the yeast genome involved. The induced transcriptional responses to the pH shift were distinctive and showed only limited resemblance to gene responses reported for other environmental stress conditions, namely increased temperature, oxidative conditions, reduced pH (succinic acid), alkaline pH and increased osmolarity.

Enzymes from extreme environments possess highly desirable traits of activity and stability under process conditions. One such example is L-aminoacylase (E.C. Number 3.5.1.14) from the thermophilic archaeon Thermococcus litoralis (TliACY), which catalyzes the hydrolysis of the amide bond between the nitrogen and the carbonyl group of an N-protected L-amino acid. As this reaction is enantiomerspecific, L-aminoacylase is often used to resolve racemic mixtures in the preparation of chiral intermediates. Using protein engineering techniques, the capabilities of such biocatalysts can be extended. This thesis seeks to compare the ability of libraries created by error-prone PCR and structure-guided mutagenesis methods to identify residues governing substrate specificity. Libraries were constructed to screen for variants which showed a shift in substrate specificity towards aliphatic amino acids, whilst maintaining the preferred benzoyl protecting group. An existing fluorescent screen for proteases was adapted for the high-throughput screening of mutant L-aminoacylase libraries, and was demonstrated to be capable of quantitatively detecting millimolar quantities of product. From an error-prone PCR library over the dimerization region of the enzyme, 10000 variants were screened against a variety of N-benzoylated amino acid substrates. Sequence alignment and homology model construction allowed a 3-D model of TliACY to be built from its closest neighbours in the PDB. Phylogenetic comparison methods were used to identify regions and residues of significance, which were then examined using sitedirected saturation mutagenesis. Purification and characterisation of selected variants of TliACY examined two mutants in detail: S4C (S100T / M106K) which exhibited a 300% improvement in catalytic efficiency over wild-type on the Nbenzoyl valine substrate, and S3B (F251K) which showed a shift in substrate preference against aliphatic amino acid substrates.

Using glycerol from biodiesel production as a fuel in a microbial fuel cell (MFC) will generate electricity and valuable by-products from what is currently considered waste. This research aims to screen E. coli (W3110, TG1, DH5, BL21), P. freudenreichii (subspecies freudenreichii and shermanii), and mixed cultures enriched from compost (AR1, AR2, AR3) as anodic biocatalysts in a glycerol-oxidizing MFC. Anaerobic fermentation experiments were performed to determine the oxidative capacity of each catalyst towards glycerol. Using an optimized medium for each strain, the highest anaerobic glycerol conversion from each group was achieved by E. coli W3110 (4.1 g/L), P. freudenreichii ssp. shermanii (10 g/L), and AR2 (20 g/L). These cultures were then tested in an MFC system. All three catalysts exhibited exoelectrogenicity. The highest power density was achieved using P. freudenreichii ssp. shermanii (14.9 mW m-2), followed by AR2 (11.7 mW m-2), and finally E. coli W3110 (9.8 mW m-2).

With the emerging interest in integrated biorefinery concepts, there is a need to identify and develop profitable product streams and ensure the utilisation of as many waste streams as possible. Early stage bioprocess development for these processes can be facilitated by the use of high throughput bioreactor platforms that enables rapid, quantitative and scalable data acquisition. This thesis aims to establish high throughput methodologies for the production and characterisation of industrial biocatalysts within an integrated biorefinery context. Specifically, the work focuses on the production of the CV2025 ω-Transaminase (CV2025 ω-TAm) in Escherichia coli BL21 (DE3) using sugar beet vinasse, a bioethanol waste stream, as a fermentation feedstock. The high throughput platform to be explored is a 24-well, controlled microbioreactor (MBR) that provides individual monitoring and control of process parameters at the well level. Initially, batch E. coli BL21 (DE3) fermentations expressing CV2025 ω-TAm were established in the controlled MBR using a synthetic medium to provide benchmark data on cell growth and enzyme expression. These cultures indicated a good degree of monitoring and control with respect to process parameters as well as culture reproducibility across the wells. Significant enhancements in relation to maximum biomass concentration (Xmax), yield of biomass on substrate (YX/S) and CV2025 ω-TAm specific activity of 3.7, 1.9 and 2.2-fold, respectively, were shown in the MBR compared to conventional shake flask system, also representing a 31-fold volumetric reduction. Optimisation of CV2025 ω-TAm production in the MBR showed that the best cell growth and enzyme titre was achieved with an early induction (6 h), 0.1 mM IPTG and 0.024 mmol IPTG gdcw-1, yielding enhancements in Xmax, YX/S and CV2025 ω-TAm specific activity of 1.04, 1.2 and 1.4-fold, respectively over the non-optimised cultures. Control of dissolved oxygen (DO) levels between 30 - 50% oxygen saturation had no significant impact on cell growth and CV2025 ω-TAm titre. Evaluation of vinasse as a fermentation feedstock for CV2025 ω-TAm production has led to several novel findings. Characterisation of vinasse showed that the feedstock comprised mainly of glycerol along with several reducing sugars, sugar alcohols, acetate, polyphenols and protein. Preliminary results showed E. coli BL21 (DE3) cell growth and CV2025 ω-TAm production were feasible in cultures using 17 to 25% (v/v) vinasse with higher concentrations demonstrating inhibitory effects. The D-galactose in vinasse was shown to facilitate auto-induction of the pQR801 plasmid leading to comparable CV2025 ω-TAm expression as obtained in IPTG-induced cultures. Assessment of different vinasse pre-processing options confirmed the relevance of the dilution step in reducing polyphenol concentrations to below inhibitory levels. Moreover, the use of pasteurised vinasse was found to be promising for large scale applications. Further medium optimisation studies in the MBR showed the benefit of supplementing vinasse with specific media components. Supplementation of diluted vinasse medium with 10 g L-1 yeast extract enabled enhancements of 2.8, 2.5, 5.4 and 3-fold in specific growth rate, Xmax, CV2025 ω-TAm volumetric and specific activity, respectively, over those achieved in non-supplemented cultures. Additionally, the CV2025 ω-TAm titre attained here represented 81% of that obtained using an optimised synthetic medium. Investigation into the metabolic preferences of E. coli BL21 (DE3) when grown on a complex carbon source like vinasse showed the sequential metabolism of D-mannitol before glycerol utilisation, which was followed by the simultaneous metabolism of glycerol, D-xylitol, D-dulcitol and acetate thereafter. Finally, scale-up of the optimal conditions for CV2025 ω-TAm production using both synthetic and vinasse-based media, from the controlled MBR to a 7.5 L stirred tank reactor (STR) was shown based on matched kLa values and specific aeration rates. Results showed a good reproducibility with respect to cell growth, substrate consumption and CV2025 ω-TAm production between the scales, representing a 769-fold volumetric scale translation. The feasibility of further intensification of CV2025 ω-TAm production in STR at higher kLa values using both synthetic and vinasse-based media was also demonstrated leading to enhancements of 1.4 and 1.9-fold in enzyme titre, respectively. Overall, this work has established high throughput methodologies for the characterisation, optimisation and scale-up of industrial biocatalyst production. The approach was demonstrated within the context of an integrated sugar beet biorefinery. However, the utility of the high throughput approach is considered generally applicable across the industrial biotechnology sector.

The experiments which are required to directly assess the operational stability of thermostable biocatalysts can be time-consuming, troublesome, and, in the context of industry, expensive. In the present work, we develop and validate two methods for quickly estimating the total turnover number (a useful indicator of lifetime productivity) of a biocatalyst for any desired operating temperature. The first method is a heuristic approach, built upon a complete mathematical derivation from first principles, in which the total turnover number can be calculated from two simple biochemical measurements. The second method relies on a single non-isothermal, continuous-mode experiment in conjunction with mathematical modeling to determine the intrinsic deactivation parameters of the biocatalyst. Both methods provide estimates of the total turnover number which are well within one order of magnitude of the values measured directly via isothermal aging tests and therefore are extremely valuable tools in terms of the amount of experimental time eliminated.

There is an important need to immobilise enzymes for use in industry, to do this I have the promising idea that by conjugating the enzyme to a hydrogel network, thus fabricating a stable biocatalytic network would be a potential method for immobilising enzymes for the manufacture of fine chemicals, this has not been done before for octapeptide systems. Hydrogels have been previously shown as a viable way of immobilising and stabilising enzymes. In this thesis the octapeptide VKVKVEVK (V is valine, K is lysine and E is glutamic acid) is used to immobilise enzymes tagged with VKVKVEVK. This peptide sequence is chosen as it forms stable hydrogels at enzyme appropriate conditions (pH 7). The enzymes chosen are; PETNR as it is well understood and is therefore a good starting point, CDH and CHMO were also chosen as they could combine with PETNR to form a cascade reaction. PETNR was both chemically conjugated to VKVKVEVK (SpepPETNR) and also genetically modified to express the peptide tag (CpepPETNR), whilst CDH and CHMO were genetically modified to express the tag (NpepCDH and CpepCHMO respectively). For S/CpepPETNR retention within the hydrogels was superior to retention for untagged PETNR. NpepCDH was found to not precipitate within the hydrogel whilst untagged was found to do so. CpepCHMO functionalised hydrogels were found to be heterogeneous. Characterisation of CpepPETNR functionalised hydrogels was undertaken using micro differential scanning calorimetry (µDSC), rheology, small angle neutron scattering (SANS) and atomic force microscopy (AFM). From the µDSC evidence of 'protective immobilisation' was observed by the increase in denaturation energy (+253 kJ mol-1) in the hydrogel in comparison to in solution (+18 kJ mol-1). The ability of S/CpepPETNR functionalised hydrogels to perform the ketoisophorone to levodione biotransformation reaction was explored with yields of 86%. S/CpepPETNR within VKVKVEVK hydrogels was found to retain ~90% conversion for at least 9 months at room temperature. Incubation overnight at 90°C resulted in a yield of 84% of levodione. These two results added more evidence for 'protective immobilisation'. Hydrogels functionalised with NpepCDH or CpepCHMO were characterised using rheology and atomic force microscopy. The biotransformation ability of NpepCDH was elucidated; the overall yield of carvone was a maximum of 54% from the hydrogel phase. NpepCDH was used alongside CpepPETNR for the cascade reaction producing dihydrocarvone in low yields; however, an improvement from 2% to 13% in yield is presented. The yield of lactone products from CpepCHMO functionalised hydrogel was low at 15%. The CpepPETNR/ CpepPETNR cascade reaction proceeded with a yield of 36%. The initial activities of CpepPETNR, NpepCDH and CpepCHMO were assayed in both solution and in gel phase using a modified method. The activities were assessed with varying conditions; temperature, pH, quantity of ethanol and incubation at high and low temperatures. Generally, it was found that immobilisation within the hydrogel phase resulted in 'protective immobilisation' against non-optimal conditions. This work will be of benefit to those who are interesting immobilising enzymes within hydrogels in the future.

PETN reductase is a robust flavin-dependent oxidoreductase which demonstrates a broad substrate spectrum constituting industrially valuable C=C double bond reductions; specifically providing an efficient route to chiral nitroalkanes. The kinetic characterisation of PETN reductase with three commercially available nitro compounds establishes their viability as substrates for the enzyme, while also identifying preferable substrate structural characteristics. PETN reductase demonstrates activity with a novel range of β,β-disubstituted and α,β-disubstituted nitroalkenes. Methyl group positioning at the substrate β- or α-carbon is demonstrated to dictate the mechanism of reduction invoked by the enzyme, while the (Z)-isomers of such substrates are shown to be preferred (product ee’s up to >99 %). The crystal structure of the enzyme complex with 1-nitrocyclohexene reveals the presence of two binding conformations, only one of which is catalytically active. A site-directed mutagenesis study involving key active site residues identified a switch of stereopreference in the T26S PETN reductase mutant and determined that Tyr 186 is not essential for the reduction of nitroalkenes in PETN reductase.

A key missing tool in the chemist’s toolbox is an effective biocatalyst for macrocyclization. Macrocycles limit the conformational flexibility of small molecules, often improving their ability to bind selectively and with high affinity to a target, making them a privileged structure in drug discovery. Resorcylic acid lactones (RALs) are a class of fungal macrocyclic polyketides that exhibit anti-cancer and anti-malarial activity among others. The thioesterases (TEs) found in the biosynthetic pathways for the zearalenone (Zea) and radicicol (Rdc) resorcylic acid lactones are responsible for macrocyclization and show promising traits as biocatalysts. These RAL TEs show the highest substrate tolerance of any polyketide thioesterase to date. These TEs can efficiently cyclize 12- 18-membered rings, 14-membered macrolactams, and amino acid containing substrates. Their robustness is evident in their ability to retain activity after lyophilization/re-suspension and in high DMSO concentrations. Furthermore, the ability of Zea and Rdc TEs to macrocyclize depsipeptide substrates illustrates the first time a polyketide synthase TE has efficiently processed a peptide-containing substrate. The unique substrate tolerance of this class of TEs shows great potential as a viable biocatalyst. Herein we describe the synthesis and enzymatic results of diverse group of substrates, with the TEs from the radicicol and zearalenone biosynthetic pathways, as well initial results on the chemoenzymatic synthesis of asperterrestide A.

Asymmetric synthesis with biocatalyst has become an increasingly interesting and cost effective manufacturing process in fine chemicals, pharmaceuticals, and agrochemical intermediates. Enoate reductases from the Old Yellow Enzyme family offer high substrate efficiency, region, stereo-, and enantioselectivity in the catalyzed biotransformations. Asymmetric reduction of activated C=C bond is one of the most widely applied synthetic tools for the potential to generate up to two stereogenic centers in one step reaction. The thesis contributed to the development and characterization of the Old Yellow Enzyme family members including NRSal from Salmonella typhimurium, YersER from Yersinia bercoviei, KYE1 from Kluyveromyces lactis, and XenA from Pseudomonas putida. We explored the possible new chemistry, gathered further understanding of enzymes functionality and biochemistry, evaluated parameters such as enzyme stability, productivity, and selectivity, and improved enzyme specificity through computational guided protein engineering method. In overall, the increasing knowledge about this Old Yellow Enzyme family together with recent advances in biotechnology renders the enoate reductases a tool of choice for industrial applications.

La utilización de enzimas como catalizadores de procesos tiene interés debido a aspectos medioambientales y a sus principales propiedades: alta selectividad y especificidad y alta capacidad catalítica bajo condiciones suaves de operación. En esta tesis doctoral se desarrolló la síntesis enzimática de un Cbz-aminopoliol, compuesto de interés terapéutico, mediante un sistema multienzimático basado en el acoplamiento de la oxidación de un aminoalcohol a su correspondiente aminoaldehído y su posterior adición aldólica a dihidroxiacetona fosfato (DHAP). Se seleccionó Cbz-etanolamina como aminoalcohol modelo. Primero se estudió la oxidación de Cbz-etanolamina a Cbz-glicinal empleando la enzima cloroperoxidasa (CPO) de Caldariomyces fumago como biocatalizador. El oxidante más adecuado para la reacción fue hidroperóxido de tert-butilo (t-BuOOH). Para minimizar la desactivación enzimática causada por la presencia de peróxido, se estudiaron diferentes estrategias de adición del mismo. El mayor rendimiento de Cbz-glicinal, de 39,1%, se alcanzó con una velocidad de adición de 3 mM/h. Además, se analizaron diferentes medios de reacción para aumentar la concentración de substrato favoreciendo la velocidad de reacción y la producción de Cbz-glicinal. Aplicando dioxano a una concentración del 5% la producción de Cbz-glicinal fue 6 veces mayor que la máxima obtenida en medio acuoso (47,6 y 7,8 mM respectivamente). Posteriormente, para aumentar la estabilidad de CPO se aplicaron técnicas de modificación química: modificaciones selectivas de grupos amino en residuos de Lys y grupos carboxilo en residuos de Asp y Glu así como entrecruzamiento y oxidación de azúcares con periodato. Se evaluó la estabilidad de las enzimas modificadas a diferentes valores de pH, temperatura y en presencia de t-BuOOH. Además se estudió el efecto de las modificaciones de CPO en la oxidación de Cbz-etanolamina. Las modificaciones de grupos carboxilo mediante adición de carbodiimida y las modificaciones resultantes de la oxidación de azúcares con periodato resultaron ser mejores catalizadores que la enzima CPO nativa en términos de estabilidad y actividad a valores elevados de pH y temperatura. A 50ºC y velocidad de adición de peróxido de 12 mM/h, los rendimientos de Cbz-glicinal aumentaron desde 16,1% (utilizando enzima nativa) a 21,5-22,1% (utilizando catalizadores modificados). Sin embargo, estos resultados no resolvieron el problema de la rápida desactivación irreversible de CPO. Buscando un mayor aumento de la estabilidad de CPO se aplicaron diferentes métodos de inmovilización: adsorción iónica, enlace covalente mediante adición de carbodiimida y enlace covalente de la enzima oxidada en geles de monoaminoetil-N-aminoetil (MANA) agarosa, así como enlace covalente en soportes Eupergit® C. Se optimizaron las condiciones de trabajo para cada método de inmovilización con el objetivo de maximizar los rendimientos de inmovilización y minimizar la desactivación enzimática durante el proceso de inmovilización. Además, se analizó la presencia de limitaciones difusionales en la catálisis con enzimas inmovilizadas, así como la estabilidad de dichas enzimas en las condiciones necesarias para la reacción de interés. El sistema enzimático más estable resultó ser el obtenido mediante enlace covalente en MANA-agarosa por adición de carbodiimida. Este catalizador se aplicó en la síntesis de Cbz-glicinal, obteniéndose una conversión más alta que la obtenida en la reacción catalizada por la enzima soluble (59,9 y 47,9%, respectivamente). Finalmente, se llevaron a cabo simultáneamente las reacciones de oxidación de Cbz-etanolamina, catalizada por CPO, y adición aldólica del aminoaldehído resultante a DHAP, catalizada por la enzima recombinante ramnulosa-1-fosfato aldolasa (RhuA) producida en Escherichia coli, obteniéndose un Cbz-aminopoliol ((3R)-5-{[(benziloxi)carbonilo]amino}-5-desoxi-1-O-fosfonopent-2-ulosa) como producto final. Se estudió el efecto de la inmovilización de ambas enzimas, la configuración del reactor y el medio de reacción con el objetivo de aumentar la producción de Cbz-aminopoliol. Aplicando ambas enzimas inmovilizadas en presencia de 5% de dioxano, dicha producción alcanzó un valor de 86,6 mM (31 g/l).
There is a growing interest for the application of the enzymes as process catalysts due to the environmental issues and the unique properties that they provide: high selectivity and specificity, and high catalytic activity while working under mild operational conditions. In this doctoral thesis, the enzymatic synthesis of the valued product Cbz-aminopolyol was achieved in a multienzymatic system by coupling the enzyme catalyzed oxidation of an amino alcohol to the corresponding amino aldehyde and enzyme catalyzed aldol addition to dihydroxyacetone phosphate (DHAP). Cbz-ethanolamine was selected as a model amino alcohol. Firstly, the enzymatic oxidation of Cbz-ethanolamine to Cbz-glycinal was studied by applying chloroperoxidase (CPO) from Caldariomyces fumago as a biocatalyst. The reaction was performed successfully using tert-butyl hydroperoxide as an oxidant. Peroxide addition strategy had to be optimized in order to minimize the peroxide dependent inactivation. Cbz-glycinal yield of 39.1% was reached when the peroxide was added at the rate of 3 mM/h. Furthermore, different reaction media were analyzed looking for the way to increase substrate concentration while favoring reaction rate and Cbz-glycinal production. Use of dioxane in the concentration of 5% resulted in 6-fold improved Cbz-glycinal production compared to the value reached in aqueous reaction (47.6 mM compared 7.8 mM, respectively). Then, with the aim of further improving of the stability of CPO, chemical modifications of CPO were carried out. Side-chain selective modifications of amino groups of Lys residues, and carboxyl groups of Asp and Glu residues, as well as crosslinking and periodate oxidation of sugar moiety were performed. The stability of modified CPOs was evaluated at different pH values, temperatures, and in the presence of tert-butyl hydroperoxide. Effect of modification of CPO on the performance of the reaction of Cbz-ethanolamine oxidation was studied as well. Those modifications that involved carboxyl groups via carbodiimide coupled method and the periodate oxidation of the sugar moiety produced better catalysts than native CPO in terms of stability and activity at elevated pH values and temperatures. At the temperature of 50ºC and peroxide addition rate of 12 mM/h, yields of Cbz-glycinal were improved from 16.1%, the value reached when using native CPO, to 21.5-22.1% when using modified CPOs. Even so, the problem of rapid irreversible inactivation of CPO by peroxides remained unsolved. Expecting a more drastic improvement in CPO stability, different immobilization methods were studied: ionic adsorption, covalent attachment by carbodiimide coupled method, and covalent attachment of oxidized enzyme on monoaminoethyl-N-aminoethyl (MANA) agarose gels as well as covalent attachment on Eupergit® C. Conditions for each immobilization method were optimized in order to maximize the immobilization yields and minimize the enzyme inactivation during the immobilization process. Then, the presence of diffusion limitations of the immobilized enzyme preparations as well as the stability of immobilized enzymes in the conditions at which the reaction of interest takes place were tested. The most stable immobilized enzyme system, covalent attachment on MANA-agarose via carbodiimide coupled method was finally applied as a biocatalyst for the synthesis of Cbz-glycinal reaching higher total conversion (59.9%) than in the reaction catalyzed by soluble enzyme (47.9%). Finally, the reaction of oxidation of Cbz-ethanolamine catalyzed by CPO was successfully coupled in one-pot reactor to the aldol addition of the amino aldehyde with DHAP catalyzed by recombinant rhamnulose-1-phosphate aldolase (RhuA) from Escherichia coli yielding Cbz-aminopolyol ((3R)-5-{[(Benzyloxy)carbonyl]amino}-5-deoxy-1-O-phosphonopent-2-ulose) as a final product. The effect of the immobilization of the enzymes, reactor configuration and reaction medium were studied in order to improve Cbz-aminopolyol production. The production of Cbz-aminopolyol, when catalyzed by immobilized enzymes in presence of 5% dioxane reached the value of 86.6 mM (31 g/l).

The use of biotransformations in organic synthesis is a small but growing field. Biotransformations can be used for many steps which prove difficult with traditional chemistry, particularly stereospecific syntheses and chiral resolutions. However, to enable the increasing use of biotransformations in chemical synthesis, a broader range of biocatalysts will be required than those of which we are currently aware. Bioprospecting and genome sequencing are increasing the awareness of Nature's library of biocatalysts. These enzymes need to be characterised for their use as biocatalysts, and where necessary modified to meet needs that may not be catered for in nature. One currently used biocatalyst is transketolase. Transketolase is an enzyme which is produced by most organisms. As a biocatalyst it is most often extracted from spinach, or produced by recombinant Escherichia coli. Transketolase carries out a stereospecific carbon-carbon bond formation, removing a 2-carbon ketol group from a ketose sugar, and adding it to the aldehyde group of the aldose sugar. It is a useful biocatalyst as it has a broad substrate specificity. Significantly it will use hydroxypyruvate as the ketol donor, thus liberating carbon dioxide and driving the reaction in a forward direction. In this thesis the transketolase-like enzyme, 1-Deoxy-D-xylulose 5-Phosphate Synthase (DOX-P Synthase), is identified as another possible biocatalyst. Significantly DOX-P Synthase catalyses the addition of a carbonyl unit, not from hydroxypyruvate, but from pyruvate, a reaction which cannot be catalysed by transketolase. This thesis therefore describes studies that aimed to produce recombinant DOX-P Synthase as a biocatalyst, and to optimise its production. Further studies aimed to characterise the properties of DOX-P Synthase, with the aim of optimising its use as a biocatalyst. Analysis of the substrate range of DOX-P Synthase could then be used to describe the set of reactions for which it would be a useful biocatalyst. Finally, this thesis describes modifications made to the enzyme, carried out by site-directed mutagenesis, and the effects on enzyme properties.

Laccases (EC 1.10.3.2) are blue multicopper oxidases that catalyse a single electron oxidation of various phenolic substrates with an associated four-electron reduction of dioxygen to water. The varied uses of laccase as a biocatalyst can be attributed to its ability to produce a free radical from a suitable substrate. Of importance to the pharmaceutical, chemical, and industrial sectors, are the laccase-catalysed reactions providing means for the synthesis of dimeric phenolics showing biological activity, including antioxidant activity. The objective of this research was to investigate the production of value-added compounds, with biological activity, via laccase-catalysed oxidation reactions. Our laboratory has access to several unique and previously unexploited culture collections obtained from extreme environments spanning the globe. Potentially novel strains were screened for the ability to produce laccases: 14 environmental isolates, of which 2 strains were fungi, 7 were streptomycetes, and 5 were non-streptomycetes, representing the rare actinomycete genera Gordonia, Rhodococcus, Mycobacterium, Amycolatopsis, and Micromonospora were screened. This is the first report of laccase production in these species. A screening protocol, using criteria specifically suited to bioprocess development, was developed to investigate variables affecting the production of laccase by the native strains. Variables investigated included different types of media (nutritional variables), pH, temperature, incubation times, aeration and agitation, salt concentrations, and the effect of inducers on laccase production by the native strains. Of the isolates investigated, actinomycete strain Micromonospora sp. 044 30-1 showed the greatest potential for the production of laccase. This strain may be novel and the role of laccase in this strain may be related to sporulation. Various growth requirements were investigated in order to optimise for maximal laccase production by strain 044 30-1. The optimal medium for laccase production was M172F medium, pH 5, supplemented with a high concentration of Cu2+ (8 mM), and 2.0% sodium chloride. Extracellular laccase production was higher than intracellular laccase production. The successful application of a Micromonospora strain in an airlift bioreactor specifically for the synthesis of laccase was demonstrated. The biocatalytic potential of the laccase from Micromonospora sp. 044 30-1 was investigated. Laccases are responsible for the formation of radicals that can react non-enzymatically with each other to form dimers or oligomers linked by C-C or C-O bonds. This study reports on laccasecatalysed oxidative reactions involving, primarily, tyrosol, monoacetyltyrosol, and to a lesser extent, reactions with totarol, 3-hydroxyanthranilic acid, and 8-hydroxyquinoline. This study reports for the first time the isolation and structure determination of novel biocatalysis reaction products, specifically the dimeric products obtained through biocatalytic reaction of monoacetyltyrosol with Trametes versicolor laccase, and Micromonospora sp. 044 30-1 laccase. The biocatalysis reaction products of the laccase-catalysed oxidation of tyrosol and monoacetylated tyrosol showed higher antioxidant activity than the parent compounds, as determined by the 2,2- diphenyl-1-picrylhydrazyl (DPPH) and low density lipoprotein (LDL) assays, showing that the dimeric derivatives of laccase-catalysed reactions with phenolic compounds have enhanced antioxidant capabilities. The biocatalysis products were also evaluated as antimicrobials and showed antimicrobial activity against E. coli, a vancomycin resistant clinical strain of Enterococcus faecium, a clinically relevant strain of Micrococcus, and Mycobacterium aurum, a strain that displays a similar antibiotic susceptibility profile to Mycobacterium tuberculosis, the causative agent of the prevalent disease tuberculosis.

Microbial fuel cells (MFCs) can generate electricity by oxidizing the organic substrates in domestic or industrial wastewater, using microorganisms as biocatalysts. The wastewater streams from the food industry based on yeasts could be directly purified and contemporary produce electricity in MFCs. However, the electron transfer mechanism between yeasts and electrodes has not been positively identified. Saccharomyces cerevisiae was implemented as biocatalyst in the anaerobic anodic compartment of an MFC using glucose as carbon source and methylene blue as electron mediator.

This project was aimed to use a purple non-sulphur bacterium, Rhodopseudomonas palustris, as a biocatalyst for hydrogen production, from the waste of biodiesel manufacturing, crude glycerol. The goal of this project was to understand the fundamentals relevant to scaling up the process and developing an off the shelf product. The first objective was to determine the ability of R. palustris to generate hydrogen by non-growing cells in comparison to that by growing cells. Similar average hydrogen production rates and energy conversion were found for both processes but a significant difference in the hydrogen yield was observed. Hydrogen production reached ~ 80 % of the theoretical maximum hydrogen yield by non-growing R. palustris, about eight-fold of that reached by growing R. palustris. The high yield suggested that it is economically appealing to use non-growing R. palustris as the biocatalyst for continuous hydrogen production. To accomplish the proposed scale-up systems, understanding its product formation kinetics is the key. It was found that the hydrogen production rate is not growth-associated and depends solely on the dry cell mass with a non-growth associated coefficient of 2.52 (Leudeking–Piret model dP/dt=2.52 X). Light is vital for hydrogen production by non-growing R. palustris, in terms of light intensity and wavelength range. It was found that excessive or insufficient light intensity may constrain the performance. Only photons of light with appropriate wavelengths can excite cytochrome bacteriochlorophyll complexes II in R. palustris to generate hydrogen. Among white LEDs, infrared LEDs, and incandescent light bulbs, at the same light intensity, infrared LEDs gave the best results in the H2 production rate and energy conversion by non-growing cells, 22.0 % ± 1.5 % higher than that with white LEDs and around 25-30 times of that by incandescent light bulbs. It was found that non-growing R. palustris can be immobilised in alginate beads to give similar H2 production rates as that by cells suspended in media. This preliminary result pointed the direction of developing an off the shelf product of immobilised non-growing R. palustris as a biocatalyst for continuous hydrogen production.

Content: Application of biocatalyst becomes an imperative due to their eco-friendly advantages. Enzymes in pretanning for unhairing, fiber opening, defleshing and bating are well reported and practiced. However, the role of enzymes as a chemical aids is less explored and consider as a secondary applications. Leather enzymes are known for its hydrolytic behavior which makes it more suitable for pretanning operations. However, typical chemical exhaustive aids acts as a vehicle for the diffusion of chemicals, whereas enzymes aids in the splitting of fibers which facilitate the diffusion of chemicals and create more functional sites for the tanning and post tanning chemicals to interact. In this research, pickled pelts are treated with acid protease and subsequently tanned using chrome tanning agent. Enzymatic treated pelts resulted in better uptake of chromium as compared to conventionally processed leathers. Similarly, after neutralization, chrome tanned leathers are treated with alkaline protease to conventional post tanning has been carried out. Enzymatic treated wet blue leathers showed high uptake of post tanning chemical, uniform dyeing and reduction in the pollution load. From the preliminary research, an interesting finding has augmented that application of enzymes at an optimized concentration, temperature, pH and time would lead to better uptake of chrome which reduces the pollution and minimization pollution load in post tanning. This study, emphasize on the application of enzymes in tanning and post tanning for higher diffusion of chemicals. Take-Away: 1. Replacement of conventional exhaustive aids using biocatalyst 2. Higher exhaustion rate of tanning and post tanning chemicals 3. Futuristic technology for sustainable leather manufacture

The lack of access to clean electricity and water in developing nations has given importance to the development of low-cost, widely applicable energy technologies. Microbial fuel cells are being explored as potential sources of clean electricity. A microbial fuel cell (MFC) is a device, in which bacteria produce electrons by oxidizing organic material, which are shuttled from the anode to the cathode, producing a current; the only byproducts of this process are respiratory waste in the form of water and carbon dioxide. Although significant advances have been made in optimizing MFCs for power output, power outputs are not always reproducible, and most importantly, MFC performance is not yet predictable under different operating conditions. These two challenges are prerequisites to the commercialization of MFC technology. In this study, a single-chamber MFC employing E.coli K12 as the biocatalyst was used to optimize power outputs and operating conditions, and demonstrate the reproducibility of MFC data. This prototype MFC was able to produce a maximum of 100 mW/m3 of reactor volume, at optimized electrode distance (2.54 cm), ionic strength of 0.5, and using a culture electrochemically activated for three generations. The data was reproducible with maximum standard errors of ± 15 mW/m3. Using this basis, a new fuel cell design was introduced, in which the anode electrode surface was increased and reactor volume was decreased. To investigate the prediction of MFC performance under different operating conditions, the new MFC model was used in a 3-level, three factor (substrate concentration, ionic strength, and medium pH) Box-Behnken experimental design. A statistical model was constructed, which could reliably predict power outputs in the MFC with less than 10% error. The statistical model optimized operating conditions in the MFC (pH 9, NaCl concentration of 15 g/L, substrate concentration of 5 g/L), corresponding to a power density of 1027 mW/m3. The effect of dimensionless quantities on MFC performance was briefly investigated: higher Schmidt values resulted in lower power densities, indicating the negative impact of increased viscosity on mass transport; all Reynolds values resulted in washout, but increases in power densities were still observed during flow regime transitions; finally, power decreased with increase Peclet values, indicating that convective mass transport was removing substrate and bacteria faster than reactions could occur. The results of this study contribute to the scale-up of MFC technology based on the prediction of MFC performance, the ability to produce repeatable results, and the demonstration of MFC performance as a function of dimensionless, scale independent parameters. This work furthers scholarship in a crucial area of MFC research, necessary for the technology's widespread application.
Le manque d'accès à l'électricité et à l'eau potable parmi les pays en développement augmente l'importance de l'innovation en domaine de technologie verte et énergie renouvelable, afin d'introduire une technologie qui est applicable à grande échelle. En tant que tel, les piles à combustible microbien sont présentement recherchées. Une pile à combustible microbien (PCM) est un appareil dans lequel les bactéries sont utilisées à oxyder les molécules organiques, afin de libérer des électrons; ces électrons sont transférés hors de la cellule à l'anode jusqu'au cathode, produisant le courant. Les seuls sous-produits de ce processus sont de l'eau et du dioxyde de charbon. Quoique le domaine de recherche en PCM ait avancé, notamment en optimisation de la production d'électricité, les puissances de sortie ne sont pas toujours reproductibles de façon fiable, et il est présentement impossible de prédire la performance des PCM aux conditions opératoires différentes. La résolution de ces deux défis est considérée parmi les questions le plus importantes de la recherche en PCM. Pendant cette étude, une PCM à un seul compartiment, à l'emploi de l'E. coli K12 comme catalyseur biologique, a été construite au but d'optimiser la production d'électricité et les conditions opératoires, et pour démontrer des données reproductibles. Ce prototype était capable de produire une puissance maximale de 100 mW/m3 (volume du réacteur), aux conditions suivants : espace de 2.54 cm entre les électrodes, force ionique de 0.5, et culture électrochimique de troisième génération. Les données étaient reproductibles avec erreur minimale (± 15 mW/m3). Étant donné ces résultats, un prototype nouveau, de moins volume, était introduit, avec un anode de graphite en format pinceau. Une plan d'expérience Box-Behnken (trois facteurs de trois niveaux chaque) était conçu afin de prédire la performance de la PCM aux conditions opératoires différentes (concentration de substrat, concentration de NaCl, et pH). Un modèle statistique était construit, capable de prédire la puissance électrique de la PCM avec erreur minimale (moins de 10%). Selon le modèle, les conditions opératoires optimales (pH 9, concentration de NaCl 15 g/L, concentration de lactose 5 g/L) ont correspondu à une puissance de 1027 mW/m3. L'effet des quantités sans dimensions sur la performance de PCM était recherché brièvement : lorsque le valeur de Sc augmentait, la puissance décroisse, indiquant l'effet négatif de la viscosité élevée sur le transport de la masse en PCM; les valeurs de Re examinés ont tous résulté en dilution extrême de la culture en PCM, mais l'accroissement de puissance été observé pendant les transitions d'un régime d'écoulement à l'autre; finalement, la puissance électrique décroissaient lorsque le valeur de Pe augmentait, un effet qui indique que la transport de masse en PCM était trop fort. Les résultats de cette étude montent les efforts en commercialisation des PCM, ayant contribué les données sur la prédiction de la performance des PCM qui sont reproductibles, et la description de relations entre la performance des PCM et les quantités sans dimension. La recherche présentée ici avance un parti crucial du domaine de PCM.

Les catalyseurs chimiques sont utilisés dans différents procédés de synthèse. Cependant, la pollution qu'ils causent sur l'environnement n’est pas prise en considération. Les procédés de synthèse chimique nécessitent généralement un grand volume de solvants organiques, produisant d’énormes quantités de déchets chimiques, souvent toxiques et non dégradables. Le remplacement des catalyseurs chimiques par des biocatalyseurs (enzymes) pourrait donc bénéficier de leur nature écologique et de leur grande sélectivité envers les produits désirés. Néanmoins, la faible activité et stabilité des enzymes ainsi que leurs coûts élevés sont des obstacles majeurs au développement des systèmes enzymatiques. Par conséquent, des études axées sur le développement de systèmes biocatalytiques plus actifs, stables et rentables, pouvant ouvrir les portes vers un environnement plus vert, sont très souhaitables. Parmi les enzymes qui catalysent des réactions d’importance dans de nombreux procédés de synthèse, le cytochrome P450 BM3 issu de Bacillus megaterium fait l'objet de cette thèse. L'enzyme est capable d’hydroxyler les liaisons C–H des acides gras (C₁₂-C₂) à température ambiante et pH physiologique. Pour cette réaction, BM3 n'a besoin que d’oxygène et de deux électrons habituellement obtenus de son cofacteur naturel, le NADPH. Cependant, pour engager cette enzyme dans les réactions d'hydroxylation, quelques obstacles importants doivent être surmontés : (i) le cofacteur coûteux (NADPH), devrait être remplacé par une source d'électrons moins chère ou régénérée, (ii) la stabilité enzymatique devrait être améliorée et (iii) l'enzyme devrait être facilement récupérable du milieu de réaction pour être réutilisée. Dans ce contexte, cette étude propose pour la première fois l'immobilisation d'un BM3 sur des nanoparticules magnétiques (NMP) d’oxyde de fer. Ce système enzymatique bénéficie (i) de la préférence de l'enzyme pour les cofacteurs NADH et BNAH (moins chers que le NADPH), (ii) de la réutilisation facile du biocatalyseur et (iii) d’une stabilité significative de l’enzyme lors du stockage. Les NMP synthétisées ont été fonctionnalisées pour permettre l’immobilisation de l'enzyme par adsorption ou liaison covalente. Par conséquent, les BM3-NMP adsorbées / réticulées ou liées de façon covalente ont été obtenues en immobilisant P450 BM3 (R966D / W1046S) sur Ni²⁺-PMIDA-NMP ou sur des NMP activés par glutaraldéhyde, respectivement.
L'activité de l’enzyme immobilisée a été comparée avec celle de l’enzyme libre dans la réaction d'hydroxylation du 10-pNCA comme substrat modèle. L'acide myristique a également été utilisé comme substrat modèle pour confirmer la capacité d’hydroxylation sélective de l’enzyme sur les atomes de carbone ω-1, -2 ou -3. Pour les mêmes conditions opératoires, le BM3 adsorbé / réticulé a montré plus de 85% de l'activité de l’enzyme libre, alors que pour les BM3-NMP liées de manière covalente cela représente 60%. La séparation facile des NMP du milieu réactionnel à l’aide d’un aimant a permis de réutiliser le système enzymatique cinq fois consécutives. Après 5 cycles de réaction, l'enzyme réticulée a conservé 100% de son activité initiale. Compte tenu que le recyclage de l’enzyme libre n’est pas faisable, ce résultat est d’une importance considérable dans les applications pratiques. De plus, la stabilité de l’enzyme pendant un mois de stockage à 4 ºC a été évaluée pour chaque système de BM3. Les résultats ont montré que l’enzyme libre n’était plus active après seulement une semaine de stockage dans ces conditions. L'enzyme réticulée n'a montré qu'une activité relative de 41% après un mois de stockage, mais pour le BM3 fixée de façon covalente, la valeur correspondante a été de 80%. La cinétique de l'hydroxylation du 10-pNCA en présence de l’enzyme libre ou immobilisée a été également étudiée. Sur la base des données expérimentales, un modèle de Hill (coefficient de Hill égal à 2) a été obtenu pour l'enzyme libre. Il a été démontré que les mêmes paramètres cinétiques sont capables de prédire le comportement du système BM3-adsorbé et BM3-réticulé dans la réaction d’hydroxylation, étant donné sa similarité avec celui de l’enzyme libre. En conclusion, les résultats de cette thèse ont montré qu'un système enzymatique actif, stable et rentable peut être obtenu en immobilisant le BM3 sur des NMP fonctionnalisées. Il bénéficie autant des avantages de l'enzyme que du support. Ainsi, l'immobilisation sur des NMP d’une enzyme spécialement conçue pour remplacer le couteux NADPH par des cofacteurs moins chers mais efficaces (NADH et BNAH) offre en même temps une amélioration significative de sa stabilité et facilite son recyclage.
MNPs have been synthesized and surface functionalized to attach the enzyme via two different methods, adsorption and covalent binding. Moreover, glutaraldehyde was used to treat the adsorbed enzyme molecules on MNPs (crosslinking-adsorption). Therefore, adsorbed, crosslinked-adsorbed, or covalently bound BM3-MNPs were obtained by immobilizing P450 BM3 on synthesized Ni²⁺-functionalized MNPs or glutaraldehyde pre-activated MNPs, respectively. The immobilized enzyme activity was compared to its free counterpart in hydroxylation reaction of 10-pNCA (10-(4-Nitrophenoxy) decanoic acid) as a substrate model. Myristic acid was also used as a substrate model to confirm the enzyme selective hydroxylation at ω-1, -2, or -3 carbon positions. The effect of cofactor (NADH and its analogue, BNAH) on the enzyme activity was also investigated. The adsorbed/crosslinked-adsorbed BM3 showed more than 85% of the free enzyme activity while the covalently bound BM3-MNPs presented 60% of the free enzyme activity under the same reaction conditions. An important feature of BM3-MNPs system is the possibility of recycling the biocatalyst. Facile separation of the magnetic nanoparticles from the reaction medium by applying a magnet provided the opportunity of reusing the enzymatic system for five times. After 5 cycles of reaction, the crosslinked-adsorbed enzyme retained 100% of its initial activity. Although the covalently bound enzyme showed, only half of the crosslinked-adsorbed enzyme activity, its storage stability was more significant. Taking into account that the enzyme reuse is an essential concern in many large-scale applications and the free BM3 cannot be recovered and reused, this result is noteworthy. Storage stability tests revealed that the free enzyme became inactive after one-week while the crosslinked-adsorbed enzyme and the covalently attached BM3 on MNPs showed 41% and 80% relative activity after one month, respectively. Finally, the steady-state kinetics of 10-pNCA hydroxylation by free and immobilized BM3 was investigated. Based on the experimental data, a non-Michaelis-Menten, Hill model (Hill coefficient of 2) was obtained for the free enzyme which could also predict the adsorbed and crosslinked-adsorbed BM3-MNPs system performance. This sigmoidal behavior was found to be independent of enzyme concentration and type of cofactor. However, since the enzyme activity was only 60% of the free enzyme for covalently bound BM3, further studies are necessary for a better understanding of this system. In summary, the results of this thesis show that an active, stable, and cost-effective BM3-MNPs system can be obtained by immobilizing an engineered BM3 on functionalized MNPs. Such systems benefit from the advantages of both enzyme and support. An engineered enzyme can fulfill the desired targets including the replacement of costly NADPH by less-expensive, yet effective cofactors namely NADH and BNAH. Furthermore, immobilization of this enzyme on MNPs improves its stability and facilitates the recycling process.
Chemical catalysts are used in different synthetic processes from lab to industrial scales. High reaction yields usually achieved by this type of processes favor their application in many industries without considering the pollution they cause to the environment. Chemical synthesis processes usually require a high volume of organic solvents and produce tons of chemical wastes which are often toxic and not degradable. Replacing conventional catalysts by biocatalysts (enzymes) can benefit from their environmentally friendly nature and high selectivity toward the desired products. Although the advantages of biocatalysts over chemical catalysts have been proven, the application of enzymes in an industrial level is still not considerable. The enzyme low activity, stability, and high cost are the main concerns in developing large-scale enzymatic systems. Therefore, in the context of a greener environment, studies focusing on the development of more active, stable, and cost-effective enzymatic systems are in great demand. Among several enzymes that can catalyze essential synthesis reactions, cytochrome P450 BM3 from Bacillus megaterium is the subject of this thesis. This enzyme hydroxylates the saturated and unsaturated C–H bonds of medium to long chain fatty acids at room temperature and physiological pH. For this reaction, BM3 only needs molecular oxygen and two electrons usually obtained from its natural cofactor, NADPH. However, to engage this enzyme in hydroxylation reactions, some important obstacles should be overcome: (i) the costly cofactor (NADPH) should be replaced by a cheaper source of electrons or regenerated, (ii) the enzyme stability should be improved, and (iii) the enzyme should be easily recovered from the reaction medium to be reused. In this context, this study proposes for the first time the immobilization of an optimized BM3 mutant on functionalized iron oxide magnetic nanoparticles (MNPs). This enzymatic system benefits from (i) the enzyme preference towards cofactors like the reasonably priced NADH and the very cheap BNAH, (ii) facile recovery and reuse of the biocatalyst (enzyme-MNPs), and (iii) the enzyme significant storage stability.

Els efectes beneficiosos que els oligosacàrids de la llet materna (OLM) confereixen a la salut dels lactants s'ha estudiat durant anys. Aquests oligosacàrids proporcionen una barrera protectora i un suport nutritiu essencials, als quals no tenen accés els nens que no prenen llet materna. La llet humana és considerada única respecte a la resta de llets de mamífers pel que fa a la quantitat i la complexitat d'oligosacàrids. Actualment, s'han identificat més de 130 estructures químiques diferents de OLM, i no es disposa de cap recurs natural que proporcioni accés a aquestes estructures tan complexes i a bastament. De la mateixa manera, la síntesi química és complicada a causa de l'estructura tan complexa i diversa que presenten els OLM, i de moment, la síntesi en gran escala no ha estat possible. La síntesi enzimàtica, en canvi, es presenta com una eina alternativa de síntesi d'aquestes molècules complexes atès que, en la naturalesa els enzims són els responsables de formar enllaços glicosídics entre carbohidrats amb alta regio- i estereoselectivitat. L'objectiu d'aquesta tesi és avaluar l'ús de l'enzim Lacto-N-biosidasa de Bifidobacterium bifidum (LnbB) com un biocatalizador eficient des de dues perspectives diferents: i) l'estudi estructural-funcional de LnbB i ii) la generació de biocatalizadors capaços de sintetitzar l'oligosacàrid d'interès (lacto-N-tetraosa) mitjançant enginyeria de proteïnes en l'enzim LnbB. En aquesta tesi, hem analitzat l'organització dels dominis dels enzims de la família GH20, i, en conseqüència, hem definit dos models d'arquitectures. El Model A conté almenys dos dominis, un domini GH20b no catalític i el GH20 catalític, que sempre es presenta acompanyat d'una α-hèlix extra. En canvi, el Model B consisteix únicament en el domini catalític GH20. Mitjançant l'expressió de diferents formes truncades de LnbB, hem descrit els requeriments estructurals per a la funcionalitat dels enzims GH20, i en particular per LnbB, per tal d’obtenir la unitat funcional mínima que conservi l'activitat enzimàtica. Respecte a la síntesi de la lacto-N-tetraosa usant com a biocatalizador noves proteïnes de LnbB obtingudes mitjançant enginyeria de proteïnes, hem contemplat dues estratègies enzimàtiques diferents. En primer lloc, l'estratègia de glicosintasa, en la qual l'enzim (amb mutació en el residu assistent) és capaç de transferir el corresponent donador activat (sucre sintètic derivat d’oxazolina) a un acceptor, sense hidròlisi del producte. En segon lloc, l'estratègia de transglicosilació millorada, en la qual, una nova generació de mutants en els llocs d'unió a l'acceptor seran capaços d'acomodar de manera més favorable un sucre en lloc d'una molècula d'aigua, i d'aquesta manera, augmentar l'activitat de transglicosilació.
Los efectos beneficiosos que los oligosacáridos de la leche materna (OLM) confieren a la salud de los lactantes se han estudiado durante años. Estos oligosacáridos proporcionan una barrera protectora y un soporte nutritivo esenciales, a los que, los niños que no toman leche materna no tienen acceso. La leche humana se considerada única respecto al resto de leches de mamíferos en cuanto a cantidad y complejidad de oligosacáridos. Actualmente, se han identificado más de 130 estructuras químicas diferentes de OLM, y no se dispone de ningún recurso natural que proporcione acceso a estas estructuras tan complejas y en cantidad suficiente. Del mismo modo, la síntesis química es complicada debido a la estructura tan compleja y diversa que presentan los OLM, y por el momento, la síntesis en gran escala no ha sido posible. La síntesis enzimática, en cambio, se presenta como una herramienta alternativa de síntesis de éstas moléculas complejas dado que, en la naturaleza las enzimas son las responsables de formar enlaces glicosídicos entre carbohidratos con alta regio- y estereoselectividad. El objetivo de esta tesis es evaluar el uso del enzima Lacto-N-biosidase de Bifidobacterium bifidum (LnbB) como un biocatalizador eficiente desde dos perspectivas diferentes: i) el estudio estructural-funcional de LnbB y ii) la generación de biocatalizadores capaces de sintetizar el oligosacárido de interés (lacto-N-tetraosa) mediante ingeniería de proteínas en el enzima LnbB. En esta tesis, hemos analizado la organización de los dominios de enzimas GH20, y, en consecuencia, hemos definido dos modelos de arquitecturas de dominio. El Modelo A contiene al menos dos dominios, un dominio GH20b no catalítico y el GH20 catalítico, que siempre se presenta acompañado de una α-hélice extra. Por el contrario, el Modelo B consiste únicamente en el dominio catalítico GH20. Mediante la expresión de diferentes formas truncadas de LnbB, hemos descrito los requerimientos estructurales para la funcionalidad de las enzimas GH20, y en particular para LnbB, de modo que se obtenga la unidad funcional mínima que conserve la actividad enzimática. Respecto a la síntesis de la lacto-N-tetraosa usando como biocatalizador nuevas proteínas de LnbB obtenidas mediante ingeniería, hemos contemplado dos estrategias enzimáticas diferentes. En primer lugar, la estrategia de glicosintasa, en la que el enzima (un mutante en el residuo asistente) es capaz de transferir el correspondiente dador activado (azúcar sintético derivado de oxazolina) a un aceptor, sin hidrólisis del producto. En segundo lugar, la estrategia de transglicosilación mejorada, en la que, una nueva generación de mutantes en los sitios de unión al aceptor serán capaces de acomodar de manera más favorable un aceptor de azúcar en lugar de una molécula de agua, y de este modo, aumentar la actividad de transglicosilación.
The potential health benefits of human milk oligosaccharides (HMO) have been studied for many years. It is well known that these oligosaccharides provide a protective barrier and nutritive support that infants with poor access to breast milk do not acquire in the first years of life. Human milk is considered to be unique among mammals in terms of the quantity and complexity of its oligosaccharides. To date, 130 chemical structures within HMO have been identified. No other natural resources provide access to these complex oligosaccharides in such large amounts, and until now, large scale synthesis of HMO has not been possible by any traditional organic chemistry methodology. Enzymatic synthesis is an alternative synthetic tool since enzymes can form the new glycosidic linkage between carbohydrates with high regio- and stereoselectivity. The objective of this thesis is to evaluate the use of Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) as an efficient biocatalyst in the following two ways: i) the structural-functional study of LnbB and ii) protein engineering of LnbB to generate biocatalysts able to synthesize the target lacto-N-tetraose. Here, we have analysed the domain organization of GH20 enzymes, and accordingly, have defined two models of domain architectures. Model A, contains at least two domains, a non-catalytic GH20b domain, and the catalytic GH20 which is always accompanied with an extra α-helix. In contrast, Model B consists only of the catalytic GH20 domain. By expressing different truncated forms of LnbB, we have described the structural requirements for functionality of GH20 enzymes, and in particular for LnbB, to obtain a minimal functional unit that retains the enzymatic activity. With regard to the synthesis of lacto-N-tetraose using new engineered LnbB proteins as biocatalysts, we envisage two different enzymatic strategies. First, the glycosynthase strategy, in which the activated donor is the corresponding synthetic sugar oxazoline and the enzyme, a mutant on the assisting residue, is able to transfer the donor to an acceptor without hydrolysis of the product. Second, the enhanced transglycosidase strategy, in which, a new generation of mutants on the acceptor subsites of the enzyme will be able to more favourably accommodate a sugar acceptor instead of water, and thus, increase transglycosylation activity.

A calcium alginate bead-immobilised biocatalyst was developed utilising the D-hydantoinase and D-N-carbamoylase from a novel, mutant Agrobacterium tumefaciens strain RU-ORPN1. The growth conditions for the inducer-independent strain were optimised for production of hydantoinase and N-carbamoylase activities. Methods for the preparation of crude enzyme extracts were evaluated in terms of hydantoinase and N-carbamoylase activities produced. After comparison of the enzyme activities and stabilities in various extracts from fresh and frozen cells, sonication of frozen cells for 5 minutes was found to be the best method for the production of the enzyme extract. The optimal pH and temperature for the hydantoinase activity were pH 10 and 30°C, respectively, while pH 9 and 40°C were optimal for Ncarbamoylase activity. The hydantoinase activity was enhanced by the addition of Mg^(2+) ions to the enzyme extract and the N-carbamoylase was enhanced by the addition of Mg^(2+), Mn^(2+) or Zn^(2+) ions to the enzyme extract. The enzyme activities increased in the presence of ATP suggesting that the enzymes may be ATP-dependent. The addition of DTT and PMSF to the enzyme extract enhanced the hydantoinase activity but had no effect on the N-carbamoylase activity. The N-carbamoylase was unstable at 40°C and was almost completely inactivated after 24 hours incubation at this temperature. The hydantoinase and N-carbamoylase appeared to be insoluble. Various techniques were investigated for the solubilisation of the enzymes including various cell lysis methods, cell lysis at extremes of pH and ionic strength, addition of a reducing agent and protease inhibitors, and treatment with hydrolysing enzymes and detergents. Treatment with Triton X-100 was most effective for the solubilisation of the enzymes indicating that the enzymes were membrane-bound. Hydropathy and transmembrane prediction plots of the predicted amino acid sequences for two identified N-carbamoylase genes from A. tumefaciens RU-ORPN1 revealed possible transmembrane regions in the amino acid sequences, and thus supported the hypothesis that the enzymes were membrane-bound. Various methods were evaluated for the immobilisation of the enzymes in whole cells and enzyme extracts. Immobilisation of the enzyme extract in calcium alginate beads was found to be the best method in terms of enzyme activity retention and stability. The hydantoinase retained 55% activity while the N-carbamoylase exhibited a remarkable sevenfold increase in activity after immobilisation by this method. Furthermore, the hydantoinase activity increased after storage at 4°C for 21 days, while the N-carbamoylase retained 30% activity after this storage period. The calcium alginate bead-immobilised enzymes were further biochemically characterised and then applied in a bioreactor system for the production of D-hydroxyphenylglycine (D-HPG) from D,L-5-hydroxyphenylhydantoin (D,L-5-HPH). The pH and temperature optima for the immobilised hydantoinase were pH 7 and 50°C, respectively, while pH 8 and 40°C were optimal for the immobilised N-carbamoylase enzyme. The immobilised enzymes showed improved thermostability at 40°C in comparison to the free enzymes and retained high levels of activity after five repeated batch reactions. Low levels of conversion were obtained in a packed-bed bioreactor containing the A. tumefaciens RU-ORPN1 biocatalyst due to the low hydantoinase activity present in the strain, relative to N-carbamoylase. A novel, packed-bed bioreactor system was therefore developed for the production of D-HPG from D,L-5-HPH using the A. tumefaciens biocatalyst in combination with a Pseudomonas sp. biocatalyst having high hydantoinase activity. A conversion yield of 22 to 30% was achieved for the production of D-HPG from D,L-5-HPH over 5 days operation demonstrating that the hydantoin-hydrolysing enzymes from A. tumefaciens RU-ORPN1 could be stabilised by immobilisation and, in combination with a biocatalyst with high hydantoinase activity, could be applied to the fully enzymatic conversion of D,L-5-HPH to D-HPG.

Treatment of industrial wastewaters contaminated with toxic and hazardous organics can be a costly process. In the case of acrylonitrile production, due to highly volatile and toxic nature of the contaminant organics, production wastewaters are currently disposed by deepwell injection without treatment. Under the terms granting deepwell injection of the waste, alternative treatments must be investigated, and an effective treatment identified. Cells of two Gram-positive bacteria, Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 were evaluated for their potential as biocatalysts for detoxification of acrylonitrile production wastewaters. Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 when multiply induced, are capable of utilizing the hazardous nitrile and amide components present in the wastewater as sole carbon and/or nitrogen sources, employing a 2-step enzymatic system involving nitrile hydratase (NHase) and amidase enzymes. There is a significant potential for overproduction of NHase upon multiple induction. However, high-level multiple induction required the presence of highly toxic nitriles and/or amides in the growth medium. Asparagine and glutamine were identified as potent inducers with overexpression at 40% of total soluble cellular protein as NHase. In native form (either cell free enzymes or whole cells) the desired NHase is very labile. In order to develop a practical catalyst to detoxify acrylonitrile production wastewaters, it is necessary to significantly improve and enhance the stability of NHase. Stabilization of desired NHase activity was achieved over a broad range of thermal and pH conditions using simultaneous immobilization and chemical stabilization. Previously where 100% of NHase activity was lost in 24 hours in the non-stabilized cells, retention of 20% of initial activity was retained over 260 days when maintained at 50-55 C, and for over 570 days for selected catalyst formulations maintained at proposed temperature of the biodetoxification process. In addition, NHase and amidase enzymes from Rhodococcus sp. DAP 96253 were purified. Cell free NHase was characterized for its substrate range and effect of common enzyme inhibitors and was compared to available information for NHase from other organisms. As a result of this research a practical alternative to the deepwell injection of acrylonitrile production wastewaters is closer to reality.

Optically pure aromatic D-amino acids, such as D-phenylalanine (D-Phe) and its derivatives, are high-value building blocks for the pharmaceutical industry. These compounds can be prepared using biocatalytic methods relying on various enzymes, including aminotransferases (ATs). ATs, also called transaminases (EC 2.6.1.X), are a subclass of pyridoxal 5′-phosphate-dependent enzymes that catalyze the transfer of the amino group from a donor substrate to a ketone acceptor. Synthesis of optically-pure amino acids using whole-cell biocatalytic cascades based on ATs possess several advantages compared to traditional chemical methods, including excellent enantioselectivity and increased process and step efficiency, which is achieved through the catalysis of multiple steps in one-pot reactions without requirement for intermediate work-ups, cofactor recycling, or toxic metals. However, enzyme biocatalysts typically need to be engineered to alter their substrate specificity or to increase their catalytic efficiency, which has limited their industrial application. Therefore, to facilitate the engineering process of ATs broadly and to produce aromatic D-amino acids, we developed a high-throughput assay for the testing of a broad range of ATs against libraries of potential substrates, and developed a biocatalytic cascade to produce optically pure aromatic D-amino acids.

Ce travail porte sur la préparation de matériaux silicatés à porosité hiérarchisée pour l'encapsulation de molécules d'intérêt dans le domaine de la pharmacie et en tant que biocatalyseur. Afin d’atteindre cet objectif, les nano-émulsions sont choisies comme empreinte pour créer les macropores du matériau en raison de la taille homogène et réduite des gouttelettes de l’émulsion (inférieure à 100 nm). Pour cela le système Remcopal 4/décane/eau est investi en déterminant les conditions les plus optimales de formation de nano-émulsion, via les méthodes d'inversion de phases. L’ajout de micelles aux nano-émulsions ne déstabilise pas les émulsions et permet la formation d’un réseau de mésopores organisés selon une symétrie hexagonale. Les matériaux hybrides issus des matériaux poreux contenant encore la phase organique sont dopés par le ketoprofène en vue d’étudier la libération de ce dernier. Celle-ci se révèle sensible au pH. De plus, cette étude de la libération du kétoprofène à partir du matériau méso-macroporeux indique qu'elle est assistée par les micelles qui sont solubilisées dans la solution réceptrice. Le deuxième objectif de ce travail est d'utiliser ces matériaux poreux en tant que biocatalyseur pour la synthèse de biodiesel à partir d'huile de colza. Pour cette application, il est nécessaire que les matériaux résistent à l’immersion dans des milieux aqueux. L’étude de la stabilité hydrothermale a montré que le matériau calciné présente la meilleure stabilité dans l’eau bouillante. Par ailleurs, le matériau peut résister jusqu’à 550°C, la structure ne subissant que des dégradations mineures. Nous avons également utilisé un matériau silicaté à double mésoporosité préparé à partir de micelles fluorées et hydrogénées coexistant dans une même solution. L'évaluation thermique et hydrothermale indique que ces matériaux présentent deux cinétiques de déstructuration qui correspondent à chacune des deux matrices ayant deux tailles de pores différents. L’immobilisation de la lipase Mml est étudiée sur le matériau méso-macroporeux calciné et sur le matériau à double mésoporosité. Les isothermes d'adsorption ont permis de mettre en évidence que le matériau à double mésoporosité peut encapsuler plus d’enzymes que son homologue méso-macroporeux. L’activité enzymatique, au regard des réactions de transestérification, est de façon inverse plus importante avec le matériau méso-macroporeux calciné
This work concerns the preparation of silica materials with hierarchical porosity for the encapsulation of molecules of interest in the field of drug delivery and as biocatalysts. In order to reach this goal, the nano-emulsions were chosen as templates for the macropores of the material because of the homogeneous and small size of the emulsion droplets (less than 100 nm). The system Remcopal 4/decane/water was investigated and the optimal conditions for which nano-emulsion is formed via the phase inversion methods were determined. Adding micelles to the nano-emulsions does not affect its stability and can form a network of mesopores organized with a hexagonal symmetry. Hybrid materials which are hierarchically porous materials where the organic phase is still present, were doped with ketoprofen to study its release, which proved to be pH sensitive. Moreover, the study of the release of ketoprofen from the meso-macroporous material indicates that it is assisted by the micelles which are solubilized in the release medium. The second objective of this work was to use these porous materials as a biocatalyst for biodiesel synthesis from colza oil. For this application it was necessary that the materials are resistant to immersion in aqueous media. The study of the hydrothermal stability shows that the calcined material has the best stability in boiling water. Moreover, the material can withstand up to 550 ° C, the structure undergoes only minor damages. We also used a dual-mesoporous silica material prepared from hydrogenated and fluorinated micelles coexisting in the same solution. Thermal and hydrothermal evaluation indicates that these materials have two different decay kinetics corresponding to each of the two matrices having different pore sizes. The immobilization of lipase Mml was studied on the meso-macroporous calcined material and the dual-mesoporous material. The adsorption isotherms were used to demonstrate that the dual-mesoporous material can encapsulate more enzymes than its meso-macroporous counterpart. On the other hand, the enzyme activity, evaluated by the transesterification reactions, is more important for the calcined meso-macroporous material

An alpha-amino ester hydrolase (AEH) applicable to synthesis of semi-synthetic antibiotics was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913. AEHs catalyze the synthesis and hydrolysis of alpha-amino beta-lactam antibiotics. The enzyme was characterized for thermodynamic and kinetic parameters. The enzyme shows optimal ampicillin hydrolytic activity at 25C and pH 6.8. The AEH enzymes have been shown to have excellent synthetic capability. Additionally, we demonstrated the first fully aqueous enzymatic one-pot synthesis of ampicillin direct from the natural product penicillin G eliminating the isolation of the intermediate 6-APA. Lastly, to improve the thermostability of the AEH a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-factors from the available crystal structures of the known AEH from Xanthomonas citri. Our best variant, which is a quadruple mutant, E143H/A275P/N186D/V622I, which has a T_50_30, the temperature at which the half-life is 30 minutes, of 34C and 1.3-fold activity compared to wild-type. Overall, we have successfully improved the understanding of the AEH class of enzymes and applied a novel cascade application, demonstrating AEHs unique applicability in the synthesis of beta-lactam antibiotics. The improved thermostability will further improve the industrial relevance of AEHs.

Addressing the low power generation of anodic biocatalysts is pertinent to the advancement of microbial fuel cell technology. While Propionibacterium freudenreichii ssp. shermanii has shown potential as a biocatalyst, its incomplete consumption of the anodic substrate is a persistent issue. This research aims to optimize substrate consumption to increase power generation using Propionibacterium freudenreichii ssp. shermanii as a biocatalyst. The effect of coculturing Geobacter sulfurreducens with Propionibacterium freudenreichii ssp. shermanii was investigated. The cocatalyst and pure culture performance was tested in an air-cathode microbial fuel cell. Geobacter sulfurreducens produced the highest maximum power density among the experimental cases. Power density produced by Propionibacterium freudenreichii ssp. shermanii was improved in the air-cathode design compared to previous experiments performed in an H-type design. The novel cocatalyst was shown to produce electricity, however a full characterization to elucidate the contribution to power generation by each microbe would be desirable to investigate.

Face à l’apparition croissante de troubles pigmentaires liés à l’exposition aux UV, le développement de nouveaux actifs blanchissants représente un enjeu majeur pour l’industrie cosmétique. De plus en plus de consommateurs s’orientent vers des produits eco-responsables, il devient urgent de développer de nouvelles méthodes de biocatalyse pour accéder à des antipigmentants. Certains glycosides, comme l’arbutine présentent des propriétés blanchissantes qui restent toutefois limitées face à l’hydrolyse rapide de la liaison O-glycosidique. L’enjeu du projet consiste à synthétiser des analogues de cette molécule en série thioglycosidique. En effet, cette liaison permet une plus grande stabilité vis à vis de l’hydrolyse. La mutation d’une glycosidase native issue de dictyoglomus thermophilum a permis d’accéder à une thioglycoligase. Cette dernière permet d’obtenir par catalyse enzymatique des analogues de l’arbutine. Six composés en série S- et O-glycosidique ont pu être synthétisés et testés en tant qu’agents dépigmentants. La méthodologie de synthèse a ensuite été appliquée pour l’obtention de thioglycolipides. Ces molécules sont connues pour leurs propriétés hydrogélifiantes permettant d’accéder à des matériaux thermoreversibles. Cinq molécules ont été identifiées en tant qu’agents hydrogélifiants. Les propriétés rhéologiques, thermiques et structurales ont été caractérisées mettant en évidence des différences significatives parmi les composés. Enfin les résultats préliminaires sur la formulation d’un produit à la fois antipigmentant et texturant s’avèrent prometteurs pour la validation d’un concept de matériau intelligent pour l’industrie cosmétique
Considering the increasing appearance of pigmentation disorders caused by UV exposure, the development of new whitening agents is a major challenge for the cosmetics industry. Consumers are turning to ecoresponsible products, it is urgent to develop new methods of biocatalysis for the access to new depigmenting agents. Some glycosides, such as arbutin, have whitening properties which are still limited because of therapid hydrolysis of the O-glycosidic bond. The challenge of this project is synthesize analogues of this molecule in thioglycoside series. Endeed, this bond allows a greater stability against the hydrolysis. The mutation of anative glycosidase to dictyoglomus thermophilum gives access to a thioglycoligase. This makes possible the enzymatic synthesis of arbutine analogues. Six molécules were synthesized and tested as depigmenting agents. The synthesis methodology was then applied to the preparation of thioglycolipids. These moleculesare known for their hydrogellating properties allowing access to thermoreversible materials. Five molecules have been identified as hydrogellating agents. The rheological, thermal and structural properties have been characterized and showed significant differences depending of the compound structure. Lastly, the preliminary results on the formulation of a product that is both antipigmenting and texturizing are promising for the validation of an smart material concept for the cosmetic industry

Optically active &
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-hydroxy ketones are important subunits of many biologically active compounds and indispensable synthons for asymmetric synthesis. Benzaldehyde Lyase from Pseudomonas fluorescens Biovar I is a novel ThDP-dependent enzyme that catalyzes the synthesis of benzoin type chiral &
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-hydroxy ketones starting from both benzaldehyde and racemic benzoin derivatives. Benzaldehyde Lyase is the first example of enzymes in the literature which leads to a chemical resolution of enantiomers of benzoin derivatives through a C-C bond cleavage reaction. Chiral 2-hydroxypropiophenone derivatives are formed by benzaldehyde lyase (BAL), catalyzing C-C bond formation after a selective C-C bond cleavage of a benzoin derivative accepted as a substrate. The enzyme uses only the (R)-benzoin derivatives as substrate for the formation of (R)-HPP derivatives and it is highly stereoselective. Thus, in the presence of the acetaldehyde as the acceptor aldehyde, the C-C bond cleavage of the benzoin molecule followed by the carboligation of the acetaldehyde to yield chiral 2-hydroxy propiophenone derivatives. Given the racemic benzoin to the enzyme as the substrate in the presence of acetaldehyde, both the racemic resolution of the substrate, revealing the unreacted (S)-Benzoin and the formation of the corresponding R-HPP occur.

Pyruvate is a valuable chemical intermediate in the production of fine chemicals used by agrochemical, pharmaceutical, and food industries. Current technology for production of pyruvic acid is based on conversion from tartaric acid and results in environmentally incompatible byproducts. An enzymatic approach to making pyruvate was developed by cloning the glycolate oxidase (GO) gene from spinach into Pichia pastoris (Payne, et al., (1995). High-level production of spinach glycolate oxidase in the methylotrophic yeast Pichia pastoris: Engineering a biocatalyst. Gene, 167(1-2), 215-219). GO is a flavoprotein (FMN dependent) which catalyzes the conversion of lactate to pyruvate with the equimolar production of hydrogen peroxide. Hydrogen peroxide can lower GO activity and make non-catalytic byproducts, so catalase was also cloned into P. pastoris to create a double transformant. Process development work was completed at the University of Iowa's Center for Biocatalysis and Bioprocessing. High-density P. pastoris fermentation (7.2 kg cells/L) was completed at the 100 L scale. Critical fermentation set-points were confirmed at 14 h glycerol feeding followed by methanol induction at 2 - 10 g/L for 30 h. After fermentation, these cells were permeabilized with benzalkonium chloride (BAC) to enable whole-cell biocatalysis and increase enzyme activity, yielding 100 U/g for GO. In 30 L enzyme reactions, permeabilized cells were recycled three times for over 92% conversion of 0.5 M lactate with an "enzyme to product" ratio of approximately 1:2 (Gough, et al., (2005). Production of pyruvate from lactate using recombinant Pichia pastoris cells as catalyst. Process Biochemistry, 40(8), 2597-2601). Though effective, the post-fermentation process for GO recovery involved several unit-operations, including multiple washing steps to remove residual BAC. The present work has focused on minimizing unit-operations by spray-drying the fermentation product to create a powdered biocatalyst. Optimal spray-drying conditions for the Buchi B-190 instrument were 150°C drying air, 15 mL/min liquid feed rate, and 600 mg cells/mL liquid feed. These conditions resulted in P. pastoris biocatalyst with activities of 80 - 100 U/g for GO and 180,000 - 220,000 U/g for catalase. The spray-dried cells retained nearly 100% of the enzyme activity compared to BAC treated cells as reported by Gough et al. Additionally, the spray-dried biocatalyst was stable at room temperature for 30 days, and no measurable enzyme leaching was observed. Then, P. pastoris was spray-dried under optimal conditions and tested for conversion of lactate to pyruvate for an improved "enzyme to product" ratio. Enzyme reaction optimization was done at the one-liter scale in DASGIP reactors. The DASGIP system contained four parallel reactors with control of temperature, pH, and dissolved oxygen. Other key variables included substrate loading, conducting the reaction in buffer or water, minimizing enzyme concentration, and maximizing the number of enzyme recycles. Optimal performance was achieved in water at pH 7.0 with an operating temperature of 25°C and 1.0 M substrate loading. Enzyme loading was at 12 g/L for the first two cycles, and subsequently, 2 - 3 g/L of fresh cells were added every alternate cycle to reach 15 cycles. Under these conditions, 75 - 95% conversion of lactate to pyruvate was accomplished for every 12 - 16 h reaction cycle. Based on these parameters, an "enzyme to product" ratio of 1:41 was achieved.

Дисертація на здобуття наукового ступеня кандидата технічних наук за спеціальністю 05.18.06 – технологія жирів, ефірних масел і парфумерно-косметичних продуктів. – Національний технічний університет "Харківський політехнічний інститут" МОН України, Харків, 2019. У дисертаційній роботі теоретично та експериментально обґрунтовано необхідність удосконалення технології виробництва жирових систем для маргаринів з мінімальним вмістом транс-ізомерів і визначено необхідні технологічні прийоми щодо її реалізації. Жирові системи є невід’ємною складовою харчування людини. Тому сучасні вимоги до підвищення якості та безпеки обумовлюють вдосконалення відповідних технологій їх виробництва. Сформована кон'юнктура зовнішнього і внутрішнього ринків диктує необхідність нарощування темпів виробництва основних олійно-жирових продуктів з високою доданою собівартістю, зокрема маргаринів. В залежності від виключення або впровадження того чи іншого компонента в рецептуру жирового продукту, покращується або значно змінюється його властивість. Необхідність корегування харчового раціону сучасної людини в напрямку зниження в ньому частки жирів, які в своєму складі містять надмірну кількість транс-ізомерів жирних кислот, обумовлена соціальним ефектом споживання жирових продуктів підвищеної якості та спрямована на оздоровлення населення України. Доцільність зниження вмісту транс-ізомерів в жирових системах ґрунтується на доведеному зв'язку між кількістю вказаних ізомерів у складі жирових харчових продуктів і підвищенням ризику розвитку серцево- судинних захворювань, а також хвороб порушення метаболізму. Зокрема, підвищений вміст транс-ізомерів блокує дію ліпопротеїдів високої щільності, ініціюючи відкладення холестеринових бляшок на стінках кровоносних судин людини та провокуючи розвиток атеросклерозу. В роботі досліджено кінетику процесів біокаталітичного переетерифікування та гліцеролізу, на яких ґрунтується технологія виробництва жирових систем зі зниженим вмістом транс-ізомерів. З використанням математичного моделювання встановлено основні закономірності кінетики біокаталітичного переетерифікування триацилгліцеринів під дією ферментного препарату Novozym 40086 та біокаталітичного гліцеролізу триацилгліцеринів, збагачених омега-3 поліненасиченими жирними кислотами. Визначено константи швидкостей прямих і зворотних реакцій, що протікають у реакційних системах, а також константи рівноваги кожної з них. На основі отриманих даних розраховано термодинамічні параметри та зроблено висновки про внесок кожної з реакцій в загальний процес у цілому. Отримані результати слугуватимуть науковим підґрунтям для встановлення технологічних параметрів виробництва жирових систем з мінімальним вмістом транс-ізомерів. Досліджено закономірності процесу біокаталітичного переетерифікування жирів під впливом препарату Novozym 40086 та встановлено раціональні параметри технології виробництва жирових систем зі зниженим вмістом транс-ізомерів. Отримано математичну модель, яка дозволяє на основі даних про компонентний склад жирової основи прогнозувати вміст твердої фази в продуктах біокаталітичного переетерифікування, що є одним із найважливіших способів оцінки їх консистенції. Розроблена модель дозволяє вирішувати зворотну задачу – виходячи з цільової консистенції жирового продукту знаходити його рецептуру. Показана можливість удосконалення складу та властивостей жирових систем зі зниженим вміст транс-ізомерів шляхом фракціонування продукту біокаталітичного переетерифікування. Розроблено математичну модель, яка дозволяє, виходячи з даних про компонентний склад олеогелів прогнозувати їх термостабільність. При цьому було застосовано методологію поверхні відклику, а визначення невідомих значень вектора параметрів здійснювалось шляхом застосування алгоритмів регресійного аналізу. Аналіз отриманих залежностей дозволив зробити висновок, що збільшення в рецептурі олеогелів вмісту бджолиного воску від 0,30 до 3,30 % мас. обумовлює зростання термостабільності. Подальше підвищення вмісту вказаного компонента не є раціональним, оскільки практично не впливає на відклик. Разом з тим при варіюванні вмісту трипальмітину від 0,30 до 3,10 % мас. спостерігається збільшення термостабільності, а при подальшому підвищенні – зменшення цього показника. Визначено раціональні значення масових часток компонентів олеогелів: вміст бджолиного воску 3,27 % мас.; вміст трипальмітину 3,07 % мас. та вміст моноацилгліцеринів 4,70 % мас. При цих значеннях досягається максимальне значення коефіцієнта термостабільності. Показано, що кількість мезофільних аеробних і факультативно-анаеробних мікроорганізмів в м’яких маргаринах на основі жирових систем зі зниженим вмістом транс-ізомерів протягом 60 діб зберігання була нижче за встановлені стандартом норми і знаходились на рівні не вище ніж 3,4·10³, тобто більш ніж на порядок меньше за нормативний показник. Крім того, у 0,01 г досліджених зразків не виявлено БГКП, а кількісні показники наявності пліснявих грибів та дріжджів також не перевищували норми. Запропоновано технологічну схему виробництва м’яких маргаринів зі зниженим вмістом транс-ізомерів жирних кислот. Наукова новизна отриманих результатів полягає у тому, що вперше: – встановлено основні закономірності перебігу біокаталітичних процесів, що лежать в основі технології виробництва жирових систем зі зниженим вмістом транс-ізомерів; – визначено раціональні параметри біокаталітичного переетерифікування жирів під дією ферментного препарату Novozym 40086; – розроблено математичну модель, яка дозволяє на основі даних про компонентний склад жирової сировини прогнозувати вміст твердої фази за різних температур в продуктах біокаталітичного переетерифікування; – встановлено кількісні залежності термостабільності олеогелів від рецептурного складу їх дисперсної фази. Практичне значення отриманих результатів для олійно-жирової галузі полягає у створенні перспективної технології жирових систем зі зниженим вмістом транс-ізомерів з використанням продуктів біокаталітичних реакцій та олеогелів. Запропоновано рецептури емульсійних продуктів на основі розроблених жирових систем. Розроблено нормативний матеріал: ТУ У 10.4 – 02071180 – 155 : 2019 на новий вид продукту – маргарин м'який "Новел" з подовженим терміном зберігання. Результати дисертаційної роботи впроваджено у навчальний процес кафедри технології жирів та продуктів бродіння Національного технічного університету "Харківський політехнічний інститут".
The thesis for a candidate of technical science degree by speciality 05.18.06 – technology of fats, essential oils and perfume-cosmetic products. – National Technical University "Kharkiv Polytechnic Institute", The Ministry of Education and Science of Ukraine, Kharkiv, 2019. In the dissertation the need of improvement of the production technology of fatty systems for margarines with minimal content of trans-isomers has been theoretically and experimentally substantiated and the necessary technological techniques for its implementation have been determined. Fatty systems are the integral part of human nutrition. Therefore, the modern demands for quality and safety require the improvement of the respective technologies for their production. The formed conjuncture of the foreign and domestic markets dictates the need to increase the production rate of major fat-and-oil products with high added cost, in particular margarines. Depending on the exclusion or introduction of a component in the formulation of the fatty product, its property is improved or significantly changed. The need to adjust the diet of modern humans in the direction of reducing the proportion of fats, which in their composition contain an excessive amount of trans-isomers of fatty acids, arises from the social effect of consumption of high quality fatty products and aims at improving the health of the population of Ukraine. The feasibility of reducing the content of trans-isomers in fatty systems is based on the proven relationship between the content of these isomers in the composition of fatty foods and the increased risk of cardiovascular disease, as well as diseases of metabolic disorders. In particular, the increased content of trans-isomers blocks the action of high density lipoproteins, initiating the deposition of cholesterol plaques on the walls of human blood vessels and provoking the development of atherosclerosis. The kinetics of the processes of biocatalytic interesterification and glycerolysis, on which the technology of production of fatty systems with reduced content of trans isomers is based, has been investigated. The basic regularities of kinetics of biocatalytic interesterification of triacylglycerols under the action of the enzymatic preparation Novozym 40086 and biocatalytic glycerolysis of triacylglycerols enriched with omega-3 polyunsaturated fatty acids have been determined using mathematical modeling. The rate constants of the forward and reverse reactions occurring in the reaction systems and the equilibrium constants of each of them have been determined. Based on the obtained data, the thermodynamic parameters have been calculated and conclusions have been made about the contribution of each of the reactions to the overall process as a whole. The results obtained will serve as the scientific basis for the determination of technological parameters for the production of fatty systems with a minimal content of trans-isomers. The regularities of the process of biocatalytic interesterification of fats under the influence of Novozym 40086 have been investigated and rational parameters of technology of production of fatty systems with reduced content of trans-isomers have been established. A mathematical model has been obtained, which allows to predict the solid phase content of biocatalytic interesterification products, which is one of the most important ways of estimating their consistency, based on data of the fat base composition. The developed model allows to solve the inverse problem – based on the target consistency of the fat product to find its formula. The possibility of improving the composition and properties of fat systems with reduced content of trans-isomers by fractionation of the product of biocatalytic interesterification has been shown. A mathematical model has been developed that allows to predict oleogels thermal stability based on the their component composition. The response surface methodology was applied, and the unknown values of the parameter vector have been determined by applying regression analysis algorithms. The analysis of the obtained dependences made it possible to conclude that the increase in the oleogel formulation of the content of beeswax from 0.30 to 3.30% of the mass. causes an increase in thermal stability. Further enhancement of the content of the specified component is not rational, as it practically does not affect the response. However, when varying the content of tripalmitin from 0.30 to 3.10% of the mass. there is an increase in thermal stability, and with a further increase – a decrease in this indicator. The rational values of the mass fractions of oleogel components have been determined: the content of beeswax is 3.27 wt%; the content of tripalmitin 3.07% of the mass. and the content of monoacylglycerols 4.70% wt. At these values, the maximum value of the coefficient of thermal stability is reached. It has been shown that the number of mesophilic aerobic and optional anaerobic microorganisms in soft margarines based on fatty systems with reduced content of trans-isomers during 60 days of storage was below the standard and were not higher than 3.4 · 103, i.e. more than an order of magnitude less than the norm. In addition, 0.01 g of the tested samples did not show CGB, and quantitative indicators of the presence of molds and yeast also did not exceed the norm. The process operational diagram for the production of soft margarines with reduced content of trans-isomers of fatty acids has been proposed. The scientific novelty of the obtained results is that for the first time: – the basic regularities of the course of biocatalytic processes underlying the technology of production of fat systems with reduced content of trans-isomers have been established; – rational parameters of biocatalytic interesterification of fats under the action of the enzyme preparation Novozym 40086 have been determined; – a mathematical model has been developed that allows to predict the content of the solid phase at different temperatures in the products of biocatalytic interesterification on the basis of data on the component composition of fatty raw materials; – quantitative dependences of thermal stability of oleogels on the formulation composition of their dispersed phase have been established. The practical significance of the results for the fat-and-oil industry consists in the developing of promising technology of fatty systems with reduced content of trans-isomers using products of biocatalytic reactions and oleogels. Formulations of emulsion products based on developed fat systems have been offered. The standard manual has been developed: TUU 10.4-02071180-087: 2019 for a new type of product – soft margarine "Novel" with extended shelf life. The results of the dissertation have been introduced into the educational process of the Department of Technology of Fats and Fermentation Products of the National Technical University "Kharkiv Polytechnic Institute".

Abstract The aim of this PhD-study was to address some of the overlapping bottlenecks in protein engineering and metagenomics by developing or applying new tools which are useful for both disciplines. Two enzymes were studied as an example: Triosephosphate Isomerase (TIM) and Uridine Phosphorylase (UP). TIM is an important enzyme of the glycolysis pathway and has been investigated via means of protein engineering, while UP is a key enzyme in the pyrimidine-salvage pathway. In this thesis TIM was used to address protein engineering aspects, while UP was used in regards to some metagenomic and bioinformatic aspects. The aspects of a structural driven rational design approach and its implications for further engineering of monomeric TIM variants are discussed. Process development based on a new technology, EnBase®, addresses the relative instability of new variants, compared to its ancestors, for further studies. EnBase® is then applied for the production of 15N isotope labeling of a monomeric TIM variant, A-TIM. Systematical function- and engineering studies on dimeric TIM and monomeric TIM in regards to the hinges of the catalytic loop-6 were conducted to investigate enzyme activity and stability. Both the A178L and P168A were proposed to induce loop-6 closure, a wanted feature for A-TIM variants. The P168A mutants are hardly active, but gave great insight into the catalytic machinery, while the A178L mutants did induce partial loop-6 closure, however in addition, monomeric A178L was destabilized. Homology driven genome mining and subsequent isolation- high throughput (HTP) overexpression of a thermostable UP from the Archaea Aeopyrum pernix was carried out as an example for the production of recombinant proteins. In addition an alternative kinetic method to study the kinetics of UP by means of NMR directly from cell lysate is discussed. The combination of expression libraries and EnBase® in a HTP manner may relieve up the gene-to-product bottleneck. The structural aspects of A. pernix UP are explored by means of simple bioinformatic tools in the last section of this thesis. A thermostable, truncated version of UP was created and its use for protein engineering in the future is explored. The long N-terminal and C-terminal ends of A. pernix UP seem to be involved in stabilizing the dimeric and hexameric structures of UP. However, deletion of the N-terminal end of A. pernix UP yielded a thermostable protein. Overall, the finding in regards to process optimization and HTP expression and optimization and the underlying methods used in the TIM studies and the UP studies are interchangeable.

O desenvolvimento sustentável é atualmente um dos maiores focos das pesquisas no mundo em virtude dos impactos ambientais, tais como aquecimento global, geração de resíduos e emissão de gases poluentes causados pelo uso de combustíveis fósseis. Deste modo, pesquisas têm sido concentradas em tecnologias que permitam a substituição de refinarias à base de petróleo por biorrefinarias que utilizam matérias-primas renováveis. Neste contexto, os fungos filamentosos surgem como um recurso promissor no desenvolvimento de novos produtos sustentáveis, entre os quais os fungos pertencentes ao filo Zigomiceto, contribuem significativamente para esse desenvolvimento e estão sendo extensivamente estudados para a aplicação em biorrefinarias, com destaque especial para os fungos do gênero Mucor. Esse gênero, particularmente da espécie Mucor circinelloides, é um potencial produtor da enzima lipase e de biomassa com quantidades significativas de lipídios (single cell oil) o que permite sua exploração no processo de produção de biodiesel. Desta forma, o objetivo deste trabalho foi explorar importantes aplicações biotecnológicas da linhagem de Mucor circinelloides URM 4182, tanto para produção de lipases quanto de lipídios. O trabalho experimental realizado consolidou dados da potencialidade do fungo para produção de lipase ligada ao micélio e sua utilização como biocatalisador para obtenção de biodiesel partir de óleos vegetais alternativos, como andiroba, coco, macaúba, palma e pinhão manso. Nas condições estudadas, foram obtidos rendimentos de transesterificação entre 88% a 97% e as conversões mais elevadas foram alcançadas para os óleos láuricos sugerindo seletividade da lipase para ácidos de cadeia curta. A potencialidade da linhagem Mucor circinelloides URM 4182 foi comprovada na obtenção de lipídios em cultivos efetuados em biorreator utilizando glicose e milhocina, respectivamente, como fontes de carbono e de nutrientes alternativos aos suplementos sintéticos. Nessas condições, valores médios de produtividade biomassa (3,10 ± 0,01 g/L/dia) contendo (31 ±0,01 % m/m) de lipídios, correspondendo produtividade lipídica de (0,97 ± 0,01 g/L.dia) foram alcançados. A composição do óleo microbiano revelou elevados teores dos ácidos graxos saturados palmítico (C16:0) e esteárico (C18:0) e dos insaturados oleico (C18:1) e linoleico (C18:2), que são os ácidos graxos semelhantes aos dos óleos vegetais convencionais utilizados para a produção de biodiesel. Além disso, foram verificados teores consideráveis do ácido gama-linolênico (GLA - C18:3), que tem relevante importância nas indústrias farmacêutica e de alimentos. A esterificação e transesterificação simultâneas do óleo microbiano ou diretamente da biomassa celular com etanol mediada por catalisadores heterogêneos bioquímicos e químicos forneceram amostras de biodiesel com elevados teores de ésteres de etila (> 97%) que atendem as normas de qualidade para uso como biocombustível.
Sustainable development is now the major research focus in the world because of the environmental impacts, such as global warming, waste generation and greenhouse gas emissions caused by the use of fossil fuels. Thus, research has been focused on technologies that enables the replacement of petroleum based refineries by biorefinery based on renewable raw materials. In this context, filamentous fungi emerge as a promising resource in the development of new sustainable products, including the fungi belonging to the phylum Zigomicete which contribute significantly to this development and are being extensively studied for using in biorefineries, with particular emphasis on the Mucor sp. fungus genus. This genus, particularly from the species of Mucor circinelloides, is a potential producer of the enzyme lipase and biomass having significant amounts of lipids (single cell oil) which allows its exploitation in the biodiesel production. Thus, the aim of this study was to explore important biotechnological applications of a Brazilian strain of Mucor circinelloides URM 4182 for both production of lipases and lipids. The experimental work consolidated this fungus capability to produce mycelium bound lipase and its use as biocatalysts for biodiesel production from alternative vegetable oils such as andiroba, coconut, macaw palm, palm and jatropha. The attained transesterification yields were in the range from 88 to 97%, and the highest conversions were achieved for lauric oils suggesting that M. circinelloides lipase has high selectivity for short chain fatty acids. The capability of this strain was also proven to produce lipids at cultivation conditions established in this work using glucose and corn steep liquor, respectively, as carbon sources and alternative nutrients to the mineral supplements. Under these conditions, average values for biomass productivity (3.10 ± 0.01 g/L/day) containing high lipis levels (31.2 ± 0.01% m/m) and lipid productivity of 0.97 ± 0.01 g/L.dia were achieved. The composition of microbial oil revealed high levels of saturated fatty acids such as palmitic acid (C16:0) and stearic (C18:0) and unsaturated oleic (C18:1) and linoleic (C18:2), which are fatty acids similar to conventional vegetable oils used for the production of biodiesel. In addition, considerable levels of gamma-linolenic acid (GLA-C18:3) were also verified which has relevant importance in the pharmaceutical and food industries. The simultaneous esterification and transesterification from microbial oil or direct fungal biomass with ethanol mediated by heterogeneous catalysts (biochemical and chemical) provided samples with high levels of ethyl esters (> 97%) that meet the quality standards for using as biofuel.

Дисертація на здобуття наукового ступеня кандидата технічних наук за спеціальністю 05.18.06 – технологія жирів, ефірних масел і парфумерно-косметичних продуктів. – Національний технічний університет "Харківський політехнічний інститут", Харків, 2019. Дисертацію присвячено удосконаленню технології жирових систем зі зниженим вмістом транс-ізомерів, що дозволяє на їх основі виробляти маргарини підвищеної харчової цінності. Встановлено основні закономірності кінетики біокаталітичного переетерифікування триацилгліцеринів під дією ферментного препарату Novozym 40086 та біокаталітичного гліцеролізу триацилгліцеринів, збагачених омега-3 поліненасиченими жирними кислотами. Визначено константи швидкостей прямих і зворотних реакцій, що протікають у реакційних системах, а також константи рівноваги кожної з них. Розраховано термодинамічні параметри біокаталітичних процесів, на яких ґрунтується технологія виробництва жирових систем зі зниженим вмістом транс-ізомерів. Встановлено раціональні значення основних параметрів біокаталітичного переетерифікування жирів під дією ферментного препарату Novozym 40086. Отримано математичну модель, яка дозволяє на основі даних про компонентний склад жирової сировини прогнозувати вміст твердої фази в продуктах біокаталітичного переетерифікування. Розроблено математичну модель, яка дозволяє, виходячи з рецептурного складу олеогелів, прогнозувати їх термостабільність. Розроблено технічні умови на новий вид м'якого маргарину зі зниженим вмістом транс-ізомерів жирних кислот. Результати роботи впроваджено у навчальний процес кафедри технології жирів та продуктів бродіння НТУ "ХПІ".
The thesis for a candidate of technical science degree by speciality 05.18.06 – technology of fats, essential oils and perfume-cosmetic products. – National Technical University "Kharkiv Polytechnic Institute", Kharkiv, 2019. Fat-based foods are an integral part of a person's daily diet. The vast majority of solid fats in the formulations of margarine products are produced by the method of partial hydrogenation, which leads to the formation of a large number of transisomers of fatty acids in their composition. The results of many studies have shown that the consumption of fats, which contain an excessive amount of trans-isomers of fatty acids, adversely affects the human body. The connection between the consumption of these fats and the increased risk of cardiovascular disease and metabolic disorders has been proved. In particular, they block the action of high-density lipoproteins, initiating the deposition of cholesterol plaques on the walls of human blood vessels, provoking the development of atherosclerosis. Therefore, solving the problem of reducing the content of trans-isomers in food is an urgent task today for the fat and oil industry of Ukraine. The dissertation is devoted to the improvement of the technology of fatty systems with reduced content of trans fatty acids, which allows to produce margarines of increased nutritional value. Based on the analyzed scientific and technical information on current trends in the production of fatty products, it has been established that the spread of the concept of healthy nutrition stimulates the demand of the population for safe products with high nutritional value. The basic regularities of kinetics of biocatalytic interesterification of triacylglycerols under the action of Novozym 40086 and biocatalytic glycerolysis of triacylglycerols enriched with omega-3 polyunsaturated fatty acids have been determined using mathematical modeling. The rate constants of the forward and reverse reactions occurring in the reaction systems and the equilibrium constants of each of them have been determined. Based on the obtained data, the thermodynamic parameters of biocatalytic processes, based on the technology of production of fat systems with reduced content of trans isomers, have been calculated. The rational values of the basic parameters of biocatalytic interesterification of fats under the action of the enzyme preparation Novozym 40086 have been established: temperature 62 °C, reaction time 368 minutes. A mathematical model has been obtained that allows to predict the solid phase content of biocatalytic interesterification products, which is one of the most important ways of estimating their consistency, based on data on the component composition of fat. The developed model allows to solve the inverse problem - based on the target consistency of the fat product to find its re-pattern. The possibility of improving the composition and properties of fatty systems with reduced content of trans-isomers by fractionation of biocatalytic interesterification product has been shown. A mathematical model has been developed that allows to predict their thermal stability based on the prescription composition of oleogels. The rational values of the mass fractions of oleogel components have been determined: the content of beeswax is 3.27 wt%; the content of tripalmitin 3.07 wt% and the content of monoacylglycerols 4.70% wt. At these values, the maximum value of the coefficient of thermal stability is reached. Soft margarines based on fatty systems with reduced trans-isomer content have been shown to have increased resistance to microbial spoilage. It has been established that the shelf life of the developed types of margarines is up to 60 days at a storage temperature of +10 ºС (conditions of the household refrigerator). The results of the work were introduced into the educational process of department of technology of fats and fermentation products of National Technical University "Kharkiv Polytechnic Institute". Specifications for a new type of soft margarine with reduced content of trans-isomers of fatty acids have been developed. The developed technology makes it possible to solve an important socioeconomic problem of providing the population with high quality fatty products. The economic feasibility of manufacturing of these products is justified by the social effect of their consumption, aimed at improving the health of population of Ukraine.

Dans le domaine de la chimie des parfums, l’optimisation des propriétés biologiques et sensorielles de substances naturelles complexes, via la biocatalyse, présente un fort intérêt. Dans un contexte de chimie durable, ces travaux de recherche sont dédiés aux développements de modifications enzymatiques de composés purs, d’extraits et d’huiles essentielles de l’industrie des arômes et parfums. Une étude bibliographique a ainsi été consacrée à la composition des matières premières naturelles, leurs propriétés et les principales voies de biosynthèse des composés présents dans les substances naturelles complexes, ainsi que quelques éléments de réglementation. Dans un premier volet, selon un procédé de chimie durable, le but a été de rendre des produits naturels plus sains tout en conservant leurs propriétés et leur « naturalité ». La détoxification en atranol et en chloroatranol de l’extrait de mousse de chêne a été effectué menant à des absolues de mousse de chêne modifiées par biocatalyse conservant leur qualité odorante. Dans le cadre d’un projet collaboratif public-privé, des protocoles de suivis de transformations biocatalytiques ont été établis et mis en œuvre. Dans un troisième volet, des méthodologies de chimie durable ont été mises à profit afin de proposer de nouveaux ingrédients grâce à l’utilisation de procédés biotechnologiques procédant selon le principe d’économie circulaire. Le caractère naturel a été conservé et la méthode a permis de réaliser des transformations fines et ciblées pour développer des facettes olfactives intéressantes
In the field of flavor and fragrance industry, the optimization of natural flavoring essential oils and extracts’ properties by biocatalysis is really interesting. In a context of sustainable chemistry, this research project is dedicated to the development of pure compounds, extracts and essential oils by enzymatic modifications. In this way, a bibliographic study has been carried out on the composition of the natural raw materials, their properties and the main biosynthetic pathways of the compounds present in the natural complex substances and some regulation elements. In the first place, according to a process of sustainable chemistry, the goal is to make healthier natural products while keeping their properties and their "naturalness". The detoxification in atranol and chloroatranol of the oak moss extract was carried out with the oak moss absolute by biocatalysis preserving their olfactory quality. Through an academic /industrial collaboration, protocols for monitoring biocatalytic transformations were established and implemented. In a third part, sustainable chemistry methodologies were used to propose new ingredients through the use of biotechnological processes based on the circular economy principle. The natural character has been preserved and the method allowed targeted transformations to develop interesting olfactory facets

L'objectif de ce travail est la mise au point de réactions de chimie « click » fluorogéniques permettant le développement de nouveaux outils chimiques et/ou la conception d'édifices bioactifs par l'assemblage des sous-unités fonctionnalisées en présence de la cible biologique. La cycloaddition 1,3-dipolaire des ylures de pyridinium a ainsi été réalisé en conditions physiologiques (H2O, pH neutre, TA), pour la mise en œuvre de réactions de couplages pouvant être biocompatibles. En effet, les ylures de pyridinium ont l'avantage d'être facilement générés in situ à partir des sels correspondants et de mener régiosélectivement, sans catalyseur métallique, à des indolizines fluorescentes après cycloaddition. Pour cela, la synthèse et la réactivité des différents partenaires de la cycloaddition ont été étudiés, tout comme l'optimisation des conditions expérimentales. Tout d'abord, deux types de dipolarophiles se caractérisant par leur caractère déficient en électrons, ont été sélectionnés: les dérivés de l'acide propiolique (amides et esters) obtenus selon une synthèse biocatalysée utilisant les lipases (CAL B), et les alcynes conjugués à des structures hétérocycliques (quinoléine, pyridine). Concernant le dipôle, à partir d'une petite famille de sels de pyridinium (diversement fonctionnalisés) ou de cycles apparentés, l'influence de la nature et de la position des substituants a été évaluée au niveau du pKa, de la réactivité (selon une réaction modèle faisant intervenir le propiolate d'éthyle), et de la fluorescence du cycloadduit. Les résultats expérimentaux, complétés par une approche de chimie théorique, ne nous permettent pas à ce jour d'expliquer la très bonne réactivité à température ambiante des dipôles portant un groupement électro-attracteur en position 4 du sel de pyridinium (sélection des sels de cyano- et acétyl-pyridinium pour nos applications). Enfin, la fonctionnalisation des précurseurs a été effectuée par l'introduction de diverses fonctions réactives dont des amides, pour obtenir une réaction de chimie « click » facilement applicable et généralisable. La preuve de concept est en cours de réalisation au laboratoire, avec la synthèse de molécules dimériques dérivées de la tacrine, pour la conception d'inhibiteurs de l'acétylcholinestérase. La seconde application porte sur la post-fonctionnalisation des indolizines et la synthèse de structures trimériques (plateforme d'assemblage fluorescente)
In the course of investigations aimed at designing fluorescent metal-free click ligations for application in bioconjugation and drug design, we turned our attention to the use of azomethine-ylides as dipoles for [3+2] cycloadditions. In this regard, pyridinium-ylides, generated in situ from suitable pyridinium salts, are of great interest due to their good reactivity with activated alkynes, such as propiolic acid derivatives or conjugated alkynes. In addition, the corresponding fluorescent indolizines are formed in a regioselective manner and any catalyst is required. From the outset, we defined a specific set of requirements for these ligations, most notably that they be biocompatible (i.e. physiological conditions, pH 7, room temperature) and that the starting reagents could be readily functionalized with the reporter group(s) or biomolecule(s) of choice. To this end, we screened a series of pyridinium salts as suitable 1,3-dipoles. The reactivity patterns observed correlate well with the pKa of the pyridinium (ylide formation) and with the electron-withdrawing character of the pyridinium ring substituent. This enabled us to identify the 4-acetyl and 4-cyano pyridinium salts suitable for ligation with propiolic esters and amides in the desired biological conditions. The synthetic strategies used to prepare the “two partners” for this [3+2] cycloaddition are described, and our preliminary results on their applications as “click” reactions are reported in particular the formation of heterodimeric tacrine derivatives (acetylcholinesterase inhibitors) or the design of fluorescent tripodal scaffold useful in chemical biology