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Cutibacterium acnes (C. acnes) (formerly known as Propionibacterium acnes) forms part of the normal resident host microflora of the skin, large intestine and oral cavity. Despite increasing evidence for the opportunistic pathogenic role of this species within intervertebral disc (IVD) herniation and associated Modic changes, C. acnes is considered a low virulence microorganism. This thesis aimed to investigate the virulence characteristics of 67 clinical C. acnes isolates belonging to phylotypes IA1, IB, II and III obtained from human herniated IVD tissue material and eight acne lesion isolates. Phenotypic isolate characterisation was undertaken using crystal violet biofilm formation assays, Galleria mellonella (G. mellonella) waxworm infection assays, exocellular enzyme production agar assays and Western blot protein analysis. Associations between phylotype grouping and site of isolation of the isolates were then investigated. Biofilm formation crystal violet assays showed that phylotype III disc tissue isolates failed to form significant biofilms. Semi-quantitative biofilm formation classification criteria identified the majority of disc tissue and acne lesion isolates as weak biofilm formers over 7-days, indicative of the slow growth rate of this species. Phylotype-specific exocellular enzyme production demonstrated that phylotype IA1and IB isolates commonly produced β-haemolysins (84% and 67%, respectively). Gelatinaseproduction was phylotype and site of isolation-specific. The highest rates of non-specific protease enzyme production were recorded for C. acnes phylotypes IA1 and III, with 100% of these isolates showing protease activity. Ubiquitous lipase production was recorded amongst all C. acnes disc tissue and acne lesion isolates tested. Comparison of waxworm larvae mortality rates between differing C. acnes phylotypes and culture preparation techniques (cell-free, heat-killed and cell-containing inoculum preparations) showed no significant effects on G. mellonella survival rates, suggesting that C. acnes does not induce significant mortality within this infection model. However, all phylotypes of C. acnespromoted G. mellonella melanisation, indicative of insect humoral immune response activation, with cell-containing cultures of C. acnes isolates 82 (IB) and 80 (III) leading to the greatest degrees of melanisation. Therefore, the G. mellonella infection model issensitive and can discriminate between the pathogenicity of C. acnes belonging to a range of phylotypes. Additionally, following Western blotting, disc tissue C. acnes samples produced CAMP factor 1 protein. Overall, site of C. acnes isolation (IVD or acne lesion) and phylotype appeared to influence phenotypic characteristics. This suggests that the role of C. acnes within lumbar disc herniation etiology should not be readily dismissed and highlights the need for further research into the applicability of anti-CAMP factor vaccines within C. acnes disc disease management.

Microbial lipases are now increasingly employed in the redesign of animal fats and vegetable oils. Commercial scale manufacture of microbial lipases, which is limited, tends to be surrounded by secrecy and much remains to be clarified in terms of optimization of fermentation conditions. C. lipolytica metabolizes cheap carbon sources (from wastes and by-products), in particular non- carbohydrate substrates, accumulating lipid during the growth. As all potent producers of lipase are also amongst the lipid-accumulating microorganisms, this yeast shows potential as a major lipase producer. The future for lipase fermentation industries look promising due to these raw material being abundant and inexpensive. Lipase activity (triolein as a substrate) and esterase activity (p-nitrophenylpalmitate as a substrate) were measured throughout this work. Different operational strategies (batch, fed-batch, chemostat and continuous transient technique) have xi been investigated aiming lipase and esterase production by C. lipolytica 1055. Nutritional parameters (carbon and nitrogen sources, limiting substrates: glucose, Tween-80 and olive oil) under steady-state conditions at different dilution rates, as well as oleic acid and olive oil square wave oscillations have been studied. Pulsing experiments with triolein, olive oil, oleic acid and Tween-80 suggest that the oleyl residue may be responsible for the induction effect in C. lipolytica 1055 lipase and esterase production. However, the low values of lipase productivities (< 1 U/mg dry weight.h) under different cultivation methods appear to limit the advantages in using C. lipolytica 1055 as a lipase producer. Extracellular esterase productivity by C. lipolytica 1055 reached 5.3 - 6.5 U/mg dry weight.h in the presence of Tween-80 under batch and fed-batch culture subjected to oleic acid input. The esterase production phase under fed-batch process was prolonged suggesting that this operation mode could be a route towards obtaining esterase by C. lipolytica 1055. For example, a two-phase bioreactor operation may be devised with high yeast cell concentration promoted by a carbohydrate waste under batch conditions in the first stage, and a high enzyme production under fed-batch operation in the second one.

The serine hydrolase (SH) enzyme superfamily is one of the largest and most diverse enzyme classes in eukaryotes and prokaryotes. The most virulent human malaria parasite Plasmodium falciparum has over 40 predicted serine hydrolases (SH). Prior investigation on a few of these have suggested their critical role in parasite biology. The majority of the SHs in P. falciparum have not been functionally characterized. Investigation of these uncharacterized SHs will provide new insights into essential features of parasite metabolism and possibly lead to new antimalarial targets. In this study, we have employed activity-based protein profiling (ABPP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to functionally characterize SHs. In our effort to profile plasmodial SHs using ABPP, we have identified a human erythrocyte SH, acylpeptide hydrolase (APEH) in the developing parasites. This finding is the first report of internalization of host hydrolytic enzyme by the parasite. Treatment of parasites with an APEH specific triazole urea inhibitor, AA74-1, caused growth inhibition in parasites with poor potency in the first replication cycle, however, the potency dramatically increased in the second cycle. We show that this unique growth inhibition profile is due to the inability of AA74-1 to inhibit parasite-internalized APEH in vivo. These findings suggest that internalization of active APEH by the parasite is essential for parasite survival. Lipases catalyze the hydrolysis of ester bonds of lipid species such as neutral lipids and phospholipids. Although roles of lipases in propagation, as well as virulence in various organisms, have been acknowledged, in P. falciparum lipases remain understudied. We combined LC-MS/MS with the SH-directed ABPP to identify lipases of SH superfamily in P. falciparum. We have identified 16 plasmodial SHs with putative lipase activity. Bioinformatics analysis of our identified lipases is consistent with our findings. We have screened a panel of various classes of SH inhibitors in a competitive ABPP. A plasmodial putative lipase was potently and specifically inhibited by human monoacylglycerol lipase inhibitor. This inhibition profile suggests it as a monoacylglycerol lipase which plays a role in releasing fatty acids from neutral lipid. This finding shows that how inhibitor screening can aid in building hypotheses on biological roles of an enzyme. Altogether, in this dissertation, we have presented a robust strategy of identifying and functionally characterizing SHs in P. falciparum, which opens the door to the discovery of new biological processes.

Milk phospholipids and non-polar lipids can form conjugations with antioxidants and starches, in the matrices of phytosomes and milk lipid-starch complexes, respectively. In addition to complexation, milk lipids also interact with starches in food matrices, leading to physiochemical changes of associated foods. Milk lipids possess neutral colour, taste, odour, natural, clean-label, and non-allergic. However, there has been little research on interactions or complexes between milk lipids and other food components when milk lipid was used as an ingredients in different food matrices. This thesis focuses on the extraction of bovine milk phospholipids from dairy products, purification of phospholipids, and preparation of vitamin C and E phytosomes in comparison with liposomes, followed by bovine milk phospholipid/triacylglycerol dispersions. Subsequently, non-polar lipids were used to prepare milk lipid-starch complexes in two kinds of food matrices: firstly bread and secondly starch gels. In the final research chapter, starch gel-stabilized milk fats were investigated in terms of lipid digestibility. The phytosomes were made by food-grade ethanol evaporation, and were verified by FTIR, DSC, UV and CI. In vitro models were used to measure the phospholipid digestibility and cellular uptake. Amylose-lipid complexes were prepared by thermal methods. The complexes were then verified by spectroscopy analysis. Starch and milk fat digestibility was determined by in vitro assays and simulated by a multi-step reaction model. The results on milk phospholipids showed that the polar heads of milk phospholipids in phytosomes interacted with hydroxyl groups of ascorbic acid resulting in the shifting of major bonds in the phosphatidyl residues in phospholipids, and therefore, milk phospholipid-based phytosomes had greater encapsulation efficiency and in vitro digestion stability than liposomes. Additionally, in opposition to triacylglycerol, milk phospholipids showed greater lipid digestibility and exhibited antioxidant activity due to differences in molecular and hydrocolloid structure. Therefore, milk phospholipids have potential application in fortification of foods including infant formulas. In contrast to milk phospholipid-based complexes, milk fats can interact and complex with corn, rice and wheat starches in food matrices. When milk fats were hydrolysed by fungal lipase, they formed starch-fatty acid complexes during baking, delaying bread firming rate and extending shelf-life due to reduced re-crystallisation of amylopectin. Hence, lipase treatment of milk fats offers a feasible way to improve both textural and physiological properties of bread. Also, milk fats-amylose conjugation can be produced by cooking at 95˚C, providing a practical method to lower the glycaemic response of starchy foods, reducing starch digestibility by 19% in corn starch, 17% in wheat starch, and 25% in rice starch. In addition to conjugation with milk fats, starch particles can also be used to stabilize milk fat emulsions. In vitro digestion showed that the lipolysis reaction speed and extent of starch gel-stabilized milk fats were two – three-fold that of milk fat dispersion, confirming the calculation by the multi-step enzymatic reaction model. This indicates the possibility to regulate lipid digestibility by designing starch-based matrices. Beginning with bovine buttermilk, membrane filtration was proposed to manufacture enriched milk phospholipid products (11 – 20 g polar lipids/100 g products), with the current, best available process efficiency. Supercritical fluid extraction was an effective, food-compatible method to produce high purity (65 – 90%) milk phospholipid products. Overall, this thesis demonstrates the structure-property-functionality relationship of milk phospholipids/lipids in several food matrices. This thesis provided novel approaches to the further use of milk lipids as functional food ingredients, such as vesicles (Chapter 4), antioxidants for infant formulas (Chapter 5), bakery product textural improvers (Chapter 6), glycaemic index reducers of starchy foods (Chapter 7), and lipophilic compound carriers using starch-stabilized milk lipid matrices (Chapter 8). Last research chapter (Chapter 9) offered practical knowledge to develop new product and processes using milk phospholipids. In future research, besides milk phospholipid neuro-functionality, milk lipid interactions with proteins and phenolic compounds should provide interesting research questions to explore.

High altitude studies have been insightful in revealing putative hypoxia tolerant genes to improve pathological and physiological conditions that involve hypoxia or ischemia. Whole-genome sequencing of Ethiopian highlanders identified LIPE (Lipase E, Hormone Sensitive Type), as an important gene that allows them to live in chronic low oxygen conditions (Udpa et al., 2014). Using Drosophila melanogaster, ubiquitous knockdown of the ortholog of this gene, hormone-sensitive lipase (Hsl), results in increased tolerance to chronic hypoxia. To elucidate the tissue specificity of the adaptive phenotype, UAS-RNAi(Hsl) line was crossed with different cell- or tissue-specific GAL4 drivers to systematically knockdown this gene, without altering its expression in the rest of the organism. There was a remarkable difference in eclosion rates when Hsl was knocked down in eclosion hormone-expressing neurons, third instar larval fat body, oenocytes, and hemocytes, constituting a four-fold increase over controls (all knockdown crosses were >87%, P

The processing of chicken feathers poses a challenge for the poultry industry. In the USA, near 9 billion chickens are slaughtered annually, which generates 5 million tons of recalcitrant feathers to manage. Enzymatic processing of feathers has promise to increase economic potential of the feathers, however, the abundant presence of lipids on feathers serve as a likely barrier. The genomes of a feather- degrading actinomycete and related microorganisms were mined for lipolytic genes candidates, which resulted in the identification of 16 active enzymes. SFK3309 from Streptomyces fradiae var. k11 was the top performing enzyme when tested against the synthetic lipase substrate p-nitrophenyl palmitate (pNPP). SFK3309 also displayed activity against feather lipids. Wax esters are the most abundant lipid class in feather lipids and a colorimetric assay was modified to perform kinetics experiments of SFK3309 against cetyl-palmitate. The Km and Kcat were calculated to be 850 μM and 11.63 s-1, respectively. Structural studies were undertaken for SFK3309, though diffracting protein crystals were unable to be produced. Thermal-shift assays suggest the presence of a hydrophobic patch on the protein surface that may interfere with protein being monodispersed in solution. Yet, some additives hold promise at stabilizing the enzyme in solution. A different lipase from S. fradiae var. k11, SFK3087, which lacked wax ester hydrolase activity, was explored using protein engineering to improve industrial characteristics of the enzyme and initiate work in expanding the substrate specificity and performance towards wax esters. The mutation Y83A increased the thermostability of SFK3087 by 3.76°C, but enzyme activity against pNPP dropped sharply. Saturation mutagenesis was performed on the serine at residue 119 and only large, aromatic residues maintained the same stability seen in wild type. The S119F mutation had a slight increase of thermostability (0.31°C), and an overall improvement in enzyme performance against pNPP, as represented by a Kcat/Km increase of 47%. A more drastic protein engineering scheme may be necessary to incorporate wax ester specificity in an enzyme. However, our work establishes feather-degrading microorganisms produce lipolytic enzymes capable of hydrolyzing wax esters and lays the foundation for further investigation on economic impact lipids have in feather processing.

Degradative enzyme activity has been shown to be elevated in many diseases including shock, diabetes, schizophrenia, several types of cancers, and coagulation disorders. The ability to measure degradative enzyme activity in whole blood and other complex samples would be useful for the development of rapid clinical diagnostics, the elucidation of disease progression, and the design of therapeutics. Current techniques to measure degradative enzyme activity require considerable amounts of time and sample processing steps. In this dissertation, a novel method for rapid detection of degradative enzyme activity, specifically protease activity, in whole blood is applied to several disease states: diabetes, schizophrenia, and premature neonates.

Fermentation and roasting of cocoa (Theobroma cacao) decrease levels of polyphenolic flavanol compounds. However, it is largely unknown how these changes in polyphenol levels caused by processing affect cocoa's anti-diabetic and anti-obesity bioactivities, such as inhibition of certain enzymes in the body. Polyphenol profiles, protein-binding abilities, presence of compounds termed oxidative polymers, and abilities to inhibit α-glucosidase, pancreatic α-amylase, lipase, and dipeptidyl peptidase-IV (DPP4) in vitro were compared between unfermented bean (UB), fermented bean (FB), unfermented liquor (UL), and fermented liquor (FL) cocoa extracts. Overall, there were significant decreases (p

Biodiesel, as well as other commercially and industrially useful lipids, can be synthesized enzymatically in lieu of other methods. Enzymatic synthesis offers improved specificity and potentially lower manufacturing costs, as nonenzymatic synthesis requires the addition of heat and expensive or dangerous reagents. Numerous microbial enzymes in the lipase family have already been shown to catalyze the synthesis of esters, including biodiesel, under hydrophobic conditions. Enzymatic synthesis of biodiesel by naturally occurring lipases is often hampered by dependence on long-chain alcohol substrates. Enzymes that can employ small alcohols (methanol or ethanol) as substrates are at an advantage because said alcohols are generally much cheaper and less toxic. We have isolated two enzymes from methicillin resistant Staphylococcus aureus that can catalyze the esterification of fatty acid to ethanol to form fatty acid ethyl esters, as they appear to do in vivo.Lipase activity, as well as other lipase-catalyzed reactions such as the synthesis of fatty acid alkyl esters, is often dependent on localization of enzyme and substrate due to the propensity of the substrates to partition in aqueous environments. Lipases have been shown to exhibit unique conformational changes when associating with lipid substrate. These changes thermodynamically enhance enzyme-lipid association, and we have sought to exploit this co-localization by mutating a lipase to recruit additional alcohol to the microenvironment of the lipid substrate. The "rebinding" effect, which has been shown to occur when multiple receptors are closely grouped, reduces diffusion of ligand (or substrate) away from the receptor cluster, increasing the equilibrium ligand concentration local to the cluster. The addition of an alcohol-binding domain derived from the Drosophila melanogaster protein LUSH to the staphylococcal lipase creates a fusion protein with superior substrate colocalization ability. As a result, the rate of fatty acid ethyl ester synthesis is enhanced, in comparison to the wild type lipase, at low ethanol concentrations.

Biodiesel, which consists of fatty acid alkyl esters, is one of the most widely adopted and successful renewable fuels. Traditional physiochemical biodiesel production methods require high cost refined feedstocks, and so alternative methods of catalysis and feedstocks have been explored. This research investigated the use of polysiloxane entrapped lipases to catalyze the production of biodiesel from low cost feedstocks. In this work, lipase from Burkholderia cepacia (lipase PS) and lipase B from Candida antarctica (CalB) were separately entrapped using sol-gel chemistry. Optimal reaction conditions for the esterification of free fatty acids by immobilized CalB with methanol were determined. Immobilized CalB and lipase PS were then used separately and in combination on a variety of model and real substrates, to assess their suitability as catalysts for biodiesel production, with yields of 97.2 wt.% fatty acid methyl esters (FAME) from a 50% free fatty acid (FFA) model substrate after a 24 h reaction time and 94.5 wt.% FAME from yellow grease. The stability of the pair of lipases over multiple uses was then examined, and the lipases were found to retain over 95% of their activity over 10 cycles of 6 h reactions. Experiments were conducted to compare the efficacy of the lipases when ethanol replaced methanol as the acyl acceptor for the esterification and transesterification reactions, as biodiesel from ethanol has superior fuel characteristics and lower environmental impact. Reactions utilizing ethanol showed similar results to methanol reactions, but with slower reaction rates. The toxicity of methanol and ethanol to each lipase’s activity was examined, and it was found that methanol caused a loss of over 90% of lipase activity to both the lipase PS and CalB when incubated for 6 h, while ethanol caused less than 50% activity loss in both cases. Adviser: Hossein Noureddini

Immobilised lipase is a promising catalyst for biodiesel production, facilitating conversion of inedible and waste oils that cannot be used with conventional inorganic catalysts. However, the relatively high cost of lipase renders it commercially unfeasible at present. A means of reducing capital and production costs of a lipase-catalysed biodiesel plant is the integration of reaction and separation steps into an extractive reactor. The aim of this thesis is to study the application of a novel extractive reactor, the Basket Impeller Column (BIC), to biodiesel production using waste cooking oil (WCO) as feedstock and a commercial immobilised lipase, Novozym 435, as catalyst. A steady-state model of the BIC was developed using Aspen Plus process simulation software. Simulations indicated that conversion increased with the square of the stirring speed, and increased linearly with number of stages. A dimensionless correlation was derived between triolein conversion in the BIC, impeller Froude number, solvent to feed ratio (S/F) and number of stages. Crude bioethanol as solvent gave higher biodiesel yields and purer raffinate than concentrated ethanol in simulations. A phase equilibrium study of organic-aqueous systems comprised of biodiesel reaction mixtures with aqueous ethanol was conducted. Equilibrium distributions of water and ethanol were closely correlated, implying strong interdependence between the polar compounds. Distribution coefficients exhibited second order step response dependency on extent of reaction, with peaks at 40-60% extents. Batch ethanolysis of WCO identified an optimum ethanol-to-oil molar ratio at the stoichiometric value of 3. Arrhenius analysis revealed a transition in apparent activation energy at 320 K. A kinetic model based on the Ping-Pong Bi Bi mechanism was developed. Significantly, addition of just 2 wt% water triggered a 90 % decline in both rate and yield. Transient water concentrations were highly oscillatory, pointing to water as an allosteric regulator of lipase. A parametric experimental study of the BIC indicated a stirring speed of 500 rpm was optimum, while adjusting S/F had only minor effects on BIC performance. Increasing solvent ethanol from 20–60 vol% led to irreversible catalyst deactivation. Yield was proportional to number of stages, due to the low Damkohler number with respect to reactants. Time-varying organic phase holdup displayed sigmoidal trends, related to the development of an emulsion phase.

Abstract: Hepatitis C virus (HCV) is a major cause of chronic liver disease, including liver steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma. It has become apparent that the targeting of lipid droplets (LDs) by the HCV core protein and the Very Low Density Lipoprotein (VLDL) secretory pathway play important roles in the HCV lifecycle. VLDL is a triacylglycerol (TG) rich lipoprotein particle that acquires the majority of its fat cargo from the preformed TG that is stored in LDs. Therefore we hypothesize that during HCV infection the VLDL assembly/secretory process would be diverted in order to support productive viral infection. This possibility is intriguing since hepatic steatosis, characterized by hepatocellular accumulation of LDs, is a common clinical finding of HCV infection and impaired VLDL assembly/secretion characterized by hypobetalipoproteinemia is associated with chronic HCV infection. We used Huh7.5/JFH-1 cell culture system to examine the relationship between HCV life cycle and VLDL secretory pathway. Using standard biochemical approaches, we have examined the key regulators of VLDL secretory pathway during HCV infection. In particular, the contribution of two putative TG lipases, arylacetamide deacetylase (AADAC) and triacylglycerol hydrolase (TGH) to the lipolytic mobilization of cellular TG stores for secretion with VLDL were examined. These studies demonstrate that the lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency for AADAC. The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Silencing of AADAC in naïve cells confirmed that endogenous AADAC indeed plays a role in the lipolysis of cellular TG stores and in the addition of lipid to nascent VLDL. TGH was absent from Huh7.5 cells and although its re-introduction to non-infected cells enhanced the mobilization of cellular TG for secretion with VLDL and VLDL production, it was not able to restore the defective cellular TG lipolysis due to AADAC deficiency in infected cells. Finally, impaired production of HCV was observed with the AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment development for the purpose of preventing and managing the disease. The most widely distributed vaccine against TB is Bacillus Calmette-Gue´rin (BCG). Apart from being ineffective in certain individuals, BCG offers only a limited timeframe of protection, is unable to serve as a booster for extending this timeframe and due to the intradermal route of administration requires costly refrigeration and syringes. LipY protein, a M. tuberculosis cell wall lipase, may play a potential role as not only a drug target but a potential vaccine antigen. LipY is known to be up-regulated during both active infection and dormancy. In a previous study, sera from TB patients had shown an IgG and IgM response against it. In this study transplastomic Lactuca sativa and Nicotiana tabacum plants were generated by transforming the chloroplasts through the particle delivery system with pLsDv-LipY and pLD-LipY vectors respectively. The vectors were flanked by the native trnI and trnA gene sequence to facilitate homologous recombination into the chloroplast genome. The vector also contained the 16S rRNA promoter, the selectable marker gene, aadA for specitinomycin resistance, the rbcL untranslated region, the LsPpsbA (PpsbA in N. tabacum) promoter, and LsTpsbA (tpsbA in N. tabacum) untranslated region. iv Site specific integration of the LipY gene into the chloroplast genome was confirmed by PCR. Homoplasmy of transplastomic plants was confirmed by Southern blot analysis. These plants showed normal growth and were fertile, producing seeds. Once germinated, these seeds did not show Mendelian segregation of the transgene. Immunoblot analysis was performed to analyze the expression of the LipY protein. A 40kDa protein was produced in E.coli, and a 25kDa protein was produced in chloroplasts; a cleaved product in chloroplasts is still valuable as an antigen for vaccine production. Future studies will include testing this chloroplast derived antigen in animal models for vaccine development.

Myzus persicae (Sülzer), commonly known as green peach aphid (GPA), utilizes its slender stylet to penetrate the plant tissues intercellularly and consume copious amounts of photoassimilates present in the phloem sap causing extensive damage to host plants. The compatible interaction between GPA and Arabidopsis thaliana enabled us to characterize plant response to aphid infestation. Upon GPA infestation, Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT4) gene modulates premature leaf senescence, which is involved in the programmed degradation of cellular components and the export of nutrients out of the senescing leaf. Senescence mechanism is utilized by plants to limit aphid growth. In addition, PAD4 provides antixenosis (deters insect settling and feeding) and antibiosis (impair aphid fecundity) against GPA and adversely impact sieve element availability to GPA. Basal expression of PAD4 contributes to antibiosis, and the GPA-induced expression of PAD4 contributes to antixenosis. Mutation in the Arabidopsis stearoyl-ACP desaturase encoding SSI2 (suppressor of SALICYLIC ACID [SA] insensitivity2) gene that results in an accelerated cell death phenotype and dwarfing, also conferred heightened antibiosis to GPA. Results of this study indicate that PAD4 is required for the ssi2-mediated enhanced antibiosis to GPA. The PAD4 protein contains conserved Ser, Asp and His residues that form the catalytic triad of many α/β fold acyl hydrolases. Arabidopsis plants expressing mutant versions of PAD4 [PAD4(S118A) and PAD4(D178A)] supported higher numbers of GPA as compared to wild type (WT) plants in no-choice tests. Furthermore, Electrical Penetration Graph (EPG) studies revealed that S118 residue in PAD4 is essential to limit GPA feeding from the sieve elements. However, the ability to deter insect settling in choice tests was not impacted by the PAD4(S118A) and PAD4(D178A) mutations, thus suggesting that PAD4s involvement in deterring insect settling and in antibiosis are determined by separate regions of PAD4. The MPL1 (MYZUS PERSICAE INDUCED LIPASE1) gene is another critical component of Arabidopsis defense against GPA. Like PAD4, MPL1 expression is induced in response to GPA infestation. However, MPL1 is required only for antibiosis and is not essential for antixenosis against GPA. EPG analysis suggests that the mpl1 mutant allele does not impact aphid feeding behavior. Since, MPL1 exhibits lipase activity, and ssi2 petiole exudates contain elevated levels of antibiosis, we propose that antibiosis to GPA requires a lipid(s), or a product thereof.

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

We have performed molecular dynamics simulation of Pseudomonas cepacia lipase (PcL) in aqueous solutions under different solution environments. We have also performed simulations of PcL with and without its structurally important Ca++ ion. Our simulations have demonstrated the movements of the helical oligopeptide 'lid' region of PcL so as to decrease or increase access to its active site cavity. We have discussed the structural integrity of the active site geometry based on root mean squared displacement of the active site residues. The structural interdependence of the lid region and the active site residues has also been established. The effect of point charges on oppositely charged residues of PcL and their effect on secondary and tertiary structure have been demonstrated. Finally, we have made predictions on the immobilization of PcL on positively charged membranes like SBA-15-NH2 based on electrostatic charge density calculations.

Obesity and related metabolic disorders have reached epidemic levels with dire public health consequences. Efforts to stem the tide focus on behavioral and pharmacological interventions. Several hypolipidemic pharmaceutical agents target endogenous lipid receptors, including the peroxisomal proliferator activated receptor alpha (PPAR alpha) and cannabinoid receptor 1 (CB1). To further the understanding of these clinically relevant receptors, we elucidated the biochemical basis of PPAR alpha activation by lipoprotein lipolysis products and the metabolic and transcriptional responses to elevated endocannabinoid signaling. PPAR alpha is activated by fatty acids and their derivatives in vitro. While several specific pathways have been implicated in the generation of PPAR alpha ligands, we focused on lipoprotein lipase mediated lipolysis of triglyceride rich lipoproteins. Fatty acids activated PPAR alpha similarly to VLDL lipolytic products. Unbound fatty acid concentration determined the extent of PPAR alpha activation. Lipolysis of VLDL, but not physiological unbound fatty acid concentrations, created the fatty acid uptake necessary to stimulate PPAR alpha. Consistent with a role for vascular lipases in the activation of PPAR alpha, administration of a lipase inhibitor (p-407) prevented PPAR alpha dependent induction of target genes in fasted mice. Apolipoprotein CIII, an endogenous inhibitor of lipoprotein lipase, regulated access to the lipoprotein pool of PPAR alpha ligands. Our results support a role for the local generation of PPAR alpha ligands by lipase activity. The endocannabinoid system regulates diverse physiological functions, including energy balance. While loss of CB1 signaling has more pronounced effects on lipid parameters than expected based on weight loss alone, direct demonstration of endocannabinoid regulation of lipid metabolism is lacking. To test the effects of endogenously produced cannabinoids on lipid metabolism, independent of alterations in food intake, we analyzed tissues from mice treated with IDFP, an organophosphorus inhibitor of endocannabinoid breakdown. IDFP administration inhibited hepatic monoacylglycerol lipase leading to elevated levels of 2-arachidonylglycerol. We found that IDFP administration caused accumulation of apoE depleted VLDL. HDL particles accumulated apoE and failed to transfer it to VLDL in vitro. Importantly, these effects were prevented by pharmacological inhibition of CB1 and absent in plasma from CB1 mice. IDFP also caused a CB1-dependent increase in hepatic triglycerides. Thus, endocannabinoids inhibit the transfer of apoE from HDL to VLDL leading to apoE depletion of triglyceride rich lipoproteins. Microarray analysis allowed us to determine the effects of IDFP on expression of genes involved in lipid metabolism and to discover novel cannabinoid responsive genes. IDFP increased expression of lipogenic and SREBP2 target genes in a CB1-dependent fashion. On a global scale, pre-administration of a CB1 antagonist prevented many of the IDFP induced alterations in gene expression. IDFP decreased expression of genes involved in amino acid metabolism and inflammation. PCR analysis of selected mRNAs confirmed several of the key array findings. Our work indicates that endocannabinoids exert a large influence on hepatic lipid metabolism independent of food intake and suggest that peripherally restricted CB1 antagonists may be of therapeutic value. Overall, these findings shed light on the endogenous mechanisms of PPAR alpha activation and the hepatic responses to Cb1 activation. This information may help guide the continuing effort to develop treatments for metabolic disease.

Egg yolk contamination of egg whites continues to be a serious problem in the egg industry. The ability of egg whites to form stable and voluminous foams is greatly inhibited by accidental yolk contamination, even at extremely small levels. Experiments were conducted to determine if lipase can regenerate the functional properties of yolk-contaminated egg whites. Treatments included control, 0.2% yolk-contamination, and 0.2% yolk-contamination that was treated with lipase and colipase and heated at 37°C for 1 hour. Lipase from Mucor meihei and colipase from porcine pancreas were added to yolk-contaminated egg white samples to target and hydrolyze the triglycerides from egg yolk. Enzymatic hydrolysis was confirmed using thin-layer chromatography. Treatment of yolk-contaminated samples with lipase, colipase and heat yielded a drastic improvement in a number of the functional properties, including the final foam volume, foam capacity, and foaming power. These functional properties showed complete restoration to control levels. However, foam stability and foam drainage levels were not statistically different from yolk-contaminated samples that had not been enzymatically treated. Enzyme treated yolk-contaminated egg whites were also tested in an angel food cake system. There were three treatments, including an uncontaminated control, a contaminated control, and a lipase and colipase treated yolk-contaminated sample. Comparison between treatments was performed by volume analysis. The enzyme treated yolk-contaminated egg whites performed similarly to uncontaminated control angel food cakes.

Developing microbial systems capable of converting low cost lipids into value added products depends on the ability to acquire substrates from the growth media. Saccharomyces cerevisiae can acquire free fatty acids from the growth media and a portion of these lipids can be converted into new lipid products. However, they cannot acquire complex lipids from the growth media unless a nonspecific lipase is included. To circumvent lipase addition, we are genetically engineering S. cerevisiae to secrete a lipase into the growth media. We selected the LIP2 gene from Yarrowia lipolytica, which encodes a nonspecific lipase. Several modifications were made to the LIP2 gene to improve processing. Results identified strains secreting the most lipase. From these results, high producing strains were inserted into an oil inducible vector. Halo assays confirmed lipase secretion, while measuring the fatty acid composition confirmed triacylglycerol breakdown, and yeast uptake of the free fatty acids released.

Burkholderia cepacia is an opportunistic pathogen that causes a variety of infections. Individuals with cystic fibrosis, a lethal autosomal-recessive disease, contract nosocomial infections of B. cepacia that are challenging to treat due to the organism’s resistance to multiple antibiotics. The mechanisms used by the bacterium to cause these infections are currently unknown and, therefore, alternative methods of treatment or prevention are difficult to establish. This project examined several B. cepacia strains and avirulent B. cepacia transconjugants to investigate potential virulence mechanisms. The data suggest that the extracellular enzymes protease, lipase, and phospholipase C do not significantly contribute to the pathogenicity of the organism, at least in the Caenorhabditis elegans model we used. Infection, not intoxication, by B. cepacia of C. elegans was responsible, but not necessarily sufficient for the nematocidal activities of all the strains studied. The results of these experiments also support the suitability of C. elegans as an in vivo model for studying the virulence factors of B. cepacia.

Non-thermal methods of reducing bacterial numbers have been studied for more than a century. A majority of this work has focused on high pressure processing (>100 MPa). The addition of carbon dioxide (CO2) at lower pressures (

Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d2, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.

Dietary cholesterol is identified as a primary risk factor in atherosclerosis, therefore reduction in the absorption of this dietary constituent would provide an important contribution in designing a plasma lipid lowering regimen. Further, a reduction in dietary cholesterol absorption within the intestinal tract would be advantageous by providing a strategy for decreasing the amount of cholesterol reaching the body's plasma compartment. The enzymatic activities of the pancreatic lipases (CEL, PL, and PLA2) within the intestinal lumen are associated with lipid absorption, and therefore provide excellent potential as target sites for modification of dietary cholesterol absorption. This dissertation examined the role of mouse pancreatic phospholipase A2 in dietary cholesterol absorption in mice by obliterating the mouse pla2 gene, thereby destroying the expression of the protein. To generate the pancreatic (or group 1B) pla2 null mouse, the murine gene for this enzyme was first isolated and characterized. The gene consists of 4 exons interspersed by 3 introns, and is contained within approximately 8.0 nucleotide kilobases. The mouse pancreatic pla2 gene is present as a single copy gene within the genome, with no pseudogenes detected. Disruption of the gene was accomplished by homologous recombination using the neoR gene inserted into exon 2 resulting in the disruption of the Ca++ binding domain of the protein. Loss of protein expression was confirmed by both immunoblot and enzymatic activity studies. Results of cholesterol absorption assays indicated no difference in cholesterol absorption between each of the three genotypes of mice (pla2 +/+, pla2 +/-, and pla2 -/-) when [14C]cholesterol was administered in a single bolus meal. Preparation of the bolus dose with cholesterol and the nonhydrolyzable phospholipid diether phosphatidylcholine resulted in a reduction of 10-18% cholesterol absorption compared to controls without regard to pla2 genotype. Administration of cholesterol in a bolus dose containi ng 1-palmitoyl-2-[14C]oleoyl-phosphatidylcholine indicated that hydrolysis of the fatty acid occurred equally, again regardless of pla2 genotype. Results of this work suggest that additional enzymes present in the intestinal lumen may compensate for loss of pancreatic pla2 by catalyzing the hydrolysis of phospholipids and thus contributing to cholesterol absorption in the pla2 null mice.