The endocannabinoid system is composed of endocannabinoids (eCBs), their cognate receptors, and their biosynthetic and catabolic enzymes. Inhibition of serine hydrolases (catabolic enzymes), such as carboxylesterases (CES), might result in the accumulation of eCBs. eCBs, such as 2-arachidonoylglycerol (2-AG), have been shown to increase or reduce inflammation via engagement with cannabinoid receptors on immune cells. This research focuses on exploring the ability of eCBs and their metabolizing enzymes to regulate inflammation. First, a negative feedback mechanism between inflammation and the eCB system was examined by identifying serine hydrolases inhibited by lipopolysaccharide (LPS) stimulation in mice. Ces2g activity was inhibited and Il6 levels were induced in the murine spleen, suggesting a role for this enzyme in an inflammatory response, possibly to limit inflammation. IL-6 did not influence 2-AG hydrolytic activity in human peripheral blood mononuclear cells (PBMCs), but monocytic MAGL was shown to be the predominant 2-AG hydrolytic enzyme in these cells. To investigate a separate mechanism by which serine hydrolases and eCBs may regulate immune responses, mice were treated with chlorpyrifos (CPF), a pesticide known to inhibit serine hydrolases, at doses that do not inhibit acetylcholinesterase in the nervous system. This research is focused on lung tissue since epidemiologic studies have linked pesticide exposures to respiratory diseases. At low doses, Ces1c (adult and neonatal mice) and Ces1d (neonatal mice) were markedly inhibited by CPF (2.5 mg/kg, 7 d, PO). Stimulation with LPS (1.25 mg/kg, IP) following the final CPF dose produced minimal differences in lung immune responses to LPS. In follow up experiments utilizing wild-type and Ces1d-/- mice, a downregulation of Ces1c mRNA in adult Ces1d-/- mice corresponded to an upregulation of Tnfa mRNA in response to LPS in CPF-treated mice. Additionally, Ces1d was found to be expressed in murine alveolar macrophages, suggesting these cells could be used to study the role of CES1 in immunity. Overall, Ces enzymes appear to play a role in immune homeostasis either through a protective mechanism or a negative feedback mechanism to control inflammation.

Carboxylesterases (CEs) are nonspecific hydrolytic enzymes and responsible for the metabolism of xenobiotics and endobiotics that contain ester bonds. There are two human CE isoforms found in liver, CES1 and CES2. In this study it is shown that the mere abundance of CES1 protein expression in human liver does not predict its biochemical activity. The human interindividual variation in CES1 activities may attribute to several mechanisms. One possibility is the presence of endogenous inhibitors in liver, arachidonic acid (AA) and 27-hydroxycholesterol (27-HC). CES1 is also expressed in human monocytes/macrophages and is proposed to catalyze the rate-limiting step of cholesterol ester mobilization in macrophages. It is of interest to determine whether CES1 can degrade the lipid mediators, 2-arachidonoylglycerol (2-AG), prostaglandin E2-1-glyceryl ester (PGE2-G), and prostaglandin F2α-1-glyceryl ester (PGF2α-G), in monocytes/macrophages and to determine if this metabolism is inhibited by organophosphate pesticide exposure.

Thesis (Doctor of Engineering in Chemical Engineering)--Cape Peninsula University of Technology, 2018. / Lignocellulosic biomass (agro-waste) has been recommended as the most promising feedstock for the production of bioalcohols, in the biofuel industry. Furthermore, agro-waste is well-known as the most abundant organic matter in the agricultural and forestry product processing industry. However, the challenge with utilizing agro-waste as a feedstock is its highly recalcitrant structure, which limits hydrolysis to convert the holocelluloses into fermentable sugars. Conventional pre-treatment methods such as dilute acid, alkaline, thermal, hot water and enzymatic, have been used in previous studies. The challenge with these conventional methods is the generation of residual toxicants during the pretreatment process, which inhibits a high bioalcohol yield, by reducing the microbial populations’ (fermenter) ability to be metabolically proficient during fermentation. Numerous studies have been developed to improve the engineered strains, which have shown to have an ability to reduce the inhibition and toxicity of the bioalcohols produced or by-products produced during pre-treatment, while enhancing the bioalcohol production. In the present study (chapter 5), evaluation of common conventional methods for the pretreatment of the mixed agro-waste, i.e. (˃45µm to <100µm) constituted by Citrus sinensis, Malus domestica peels, corn cobs from Zea mays and Quercus robur (oak) yard waste without a pre-rinsing step at a ratio of 1:1 at 25% (w/w) for each waste material, was undertaken, focusing on hot water pre treatment followed by dilute acid (H2SO4) pre-treatment. To further pretreat the mixed agro-waste residue, cellulases were used to further hydrolyse the pre-treated agro-waste in a single pot (batch) multi-reaction process. The TRS concentration of 0.12, 1.43 and 3.22 g/L was achieved with hot water, dilute acid and cellulases hydrolysis as sequential pretreatment steps, respectively, in a single pot multi-reaction system. Furthermore, a commercial strain was used to ascertain low (C1 to C3) and high carbon content (C4+) bioalcohol production under aerobic conditions. Multiple bioproducts were obtained within 48 to 72 h, including bioethanol and 1-Butanol, 3-methyl, which were major products for this study. However, undesirable bio-compounds such as phenolics, were detected post fermentation. Since multiple process units characterised by chemical usage and high energy intensivity have been utilized to overcome delignification and cellulolysis, a sustainable, environmental benign pretreatment process was proposed using N. mirabilis “monkey cup” fluids (extracts) to also reduce fermenter inhibitors from the delignification of mixed agrowaste; a process with minimal thermo physical chemical inputs for which a single pot multi-reaction system strategy was used. Nepenthes mirabilis extracts shown to have ligninolytic, cellulolytic and xylanolytic activities, were used as an enzyme cocktail to pretreat mixed agro-waste, subsequent to the furtherance of TRS production from the agro-waste, by further using cellulase for further hydrolysis. N. mirabilis pod extracts were determined to contained carboxylesterases (529.41±30.50 U/L), β-glucosidases (251.94±11.48 U/L) and xylanases (36.09±18.04 U/L), constituting an enzymatic cocktail with a significant potential for the reduction in total residual phenolic compounds (TRPCs). Furthermore, the results indicated that maximum concentration of TRS obtainable was 310±5.19 mg/L within 168 h, while the TRPCs were reduced from 6.25±0.18 to 4.26 ±0.09 mg/L, which was lower than that observed when conventional methods were used. Overall N. mirabilis extracts were demonstrated to have an ability to support biocatalytic processes for the conversion of agro-waste to produce fermentable TRS in a single unit facilitating multiple reactions with minimised interference with cellulase hydrolysis. Therefore, the digestive enzymes in N. mirabilis pods can be used in an integrated system for a second generation biorefinery.

La tuberculose reste l'une des maladies les plus meurtrières dans le monde et de nouvelles stratégies sont urgemment demandées pour combattre Mycobacterium tuberculosis (Mtb), l'agent éthiologique de la maladie. Les lipides jouent un rôle important dans le cycle de vie de la bactérie et sont largement présents dans sa membrane et son cytoplasme, où ils peuvent servir en tant que sources de carbone et d'énergie pour favoriser la pathogénicité et la survie pendant les phases d'infection et de persistance. Dans ce contexte, les rôles des enzymes lipolytiques restent mal définis et demandent à être davantage caractérisés. La première partie de ce travail de thèse a été consacrée à l'étude des douze enzymes de Mtb homologues à la lipase hormono-sensible humaine. Les effets du MmPPOX, un composé oxadiazolone très sélectif de cette famille de protéines, ont été évalués sur les enzymes recombinantes et directement in vivo sur Mtb et M. bovis BCG. Cet inhibiteur a démontré une activité antimycobactérienne, suggérant des rôles métaboliques importants pour ces enzymes. La seconde partie de ce travail a été consacrée à l'étude des mécanismes physico-chimiques dont dépendent fortement les inhibitions des lipases et des carboxylestérases in vivo, comme la présence de substrats et/ou de détergents. La spectrométrie de masse a également été introduite en tant qu'outil rapide et puissant pour caractériser les adduits [enzyme-inhibiteur]. Enfin, nous avons développé une approche de chimie protéomique pour capturer sélectivement des hydrolases à sérine à partir de milieux biologiques complexes. / Tuberculosis remains one of the deadliest diseases in the world and new strategies are urgently needed to combat Mycobacterium tuberculosis (Mtb), its etiologic agent. Lipids play an important part in the lifetime of the bacterium, as they are widely present in the membrane and stored in the cytoplasm, where they could be used as carbon and energy sources to promote pathogenicity and survival during infection and persistence. In this context, roles of lipolytic enzymes are still poorly understood and remain to be characterized. The first part of my work was devoted to the study of twelve Mtb enzymes homologous to the human hormone-sensitive lipase. Effects of MmPPOX, an oxadiazolone compound highly selective for this family of proteins, were investigated using recombinant enzymes and directly tested in vivo using Mtb and M. bovis BCG. This inhibitor demonstrated antimycobacterial activities, suggesting important metabolic roles for these enzymes. The second part of this work was devoted to the study of physico-chemical mechanisms on which lipase and carboxylesterase inhibition could strongly depend in vivo, like presence of substrates and/or detergents. Mass spectrometry was also introduced as a direct and powerful tool to characterize [enzyme-inhibitor] adducts. Finally, we developed chemical proteomics approaches to specifically capture serine hydrolases from complex biological media. We aimed to synthesize a grafted alkylphosphonate inhibitor on a solid support by assaying several grafting strategies and matrices of various chemical natures.

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