This thesis investigates an application of immobilized lipase for pre-treating wastewater containing fats and oils, which is difficult to treat practically. The kinetics of soluble lipase was studied for establishing background of the lipase. The immobilization of lipase was adopted in order to repeatedly use the expensive lipase. The developed immobilization methods were based on the characteristics of carriers, but covalent bonding of lipase was preferred because of strong adsorption nature. Three types of materials, nylon membrane and polystyrene-divinylbenzene and silica gel beads, were used for studying the lipase immobilization characteristics. The lipase from Canada rugosa was chosen because of its relatively high catalytic activity and commercial availability. The oily wastewater sources used were a simulated mixture of olive oil and distilled water as well as actual restaurant oily wastewater. A packed bed reactor packed with immobilized lipase was suitable for the study. Moreover, a comparative study of anaerobic digestion of lipase treated and un-treated oily wastewater was undertaken to evaluate the efficiency of the lipase pre-treatment method due to lack of the relevant literature in the enzymatic wastewater treatment field. The kinetics of lipase catalyzed hydrolysis reactions was investigated in a stirred tank reactor. The experimental results confirmed that the lipase catalyzed reaction obeyed Michaelis-Menten model. The optimal pH and temperature of the lipase catalysed hydrolysis reaction were 7 and 37°C, respectively. The conversion of oil to fatty acid was dependent on the reaction time and mass of the enzyme used. The lipase activities depended on the concentrations of some selected additives. Calcium ion improved lipase activity significantly amongst the additives used. / The immobilization of lipase was carried out using different materials, nylon membranes, polystyrene-divinylbenzene beads, and silica gel. Covalent adsorption was simple and successful for immobilizing the lipase onto nylon membrane which was pre-treated with HC1 solution for releasing amino groups. The adsorption of lipase was completed after only a 2-hour reaction time. It was much more practical for this shorter adsorption time (2 hours) rather than the 24 hours required for physical capillary adsorption of lipase. The properties of the immobilized lipase and the performance of the reactors we compared amongst the soluble and immobilized lipase forms. The immobilization, particularly for covalent bonding, made lipase more resistant to thermal deactivation. It was evident that the optimum temperature was shifted from 37°C for the soluble lipase to 45 and 40°C for immobilized lipase adsorbed onto nylon and polystyrene-divinylbenzene beads, respectively. The immobilized lipase could be used repeatedly with only little activity loss. The repeatedly operational stability made the reuse of the immobilized lipase possible. Comparison was also made between two types of beads, polystyrene-divinylbenzene beads and silica gels. Though polystyrene-divinylbenzene beads showed higher lipase activity and shorter adsorption time when compared to silica gels, the forme beads were not suggested for large scale study because of high cost of the beads. On improvement achieved in this work was that the 24 hours required for silanization of silica gel was reduced to only a few hours using evaporating 3-APTES in acetone instead of refluxing 3-APTES in toluene. / It is worthwhile to point out that much higher enzyme activity was obtained using the packed bed reactor as against the membrane reactor when aqueous oil emulsion was fed into the reactors. The lipase activity was 64.2% of soluble lipase activity for the immobilized lipase in the packed reactor but its activity was hardly detectable in the membrane reactor. Moreover, the operation of the packed bed reactor solved the of separating problem that severely hampered the lipase catalytic activity in the membrane reactor in aqueous phase. This result suggests that the packed bed reacts with the immobilized lipase is applicable in treating oily wastewater. The intrinsic parameters, Vmax and Km, were evaluated to study the internal diffusional effects of the porous spherical silica gel on the immobilized lipase. The changes of Vmax and Km for the immobilized lipase from those of the soluble lipase indicated that some alteration in the lipase intrinsic properties was caused by the immobilization of lipase. However, the magnitude of Thiele modulus suggested the immobilized lipase was most likely reaction controlling. In addition, good agreement for Vmax and Km from experiments and numerical model estimations seemed to suggest that the numerical model could be used for estimating Vmax and Km for the immobilized lipase. / An application was tried for conducting the hydrolysis of oily restaurant wastewater by soluble and the immobilized lipase. Enzyme activity of both forms was severely inhibited by the oily wastewater. The enzymatic activity was only 20% and 15% for soluble and the immobilized lipase, respectively, when compared to the initial activity value for the hydrolysis of olive oil by soluble lipase. Evaluation of the efficiency for the proposed lipase pre-treatment method was carried out by monitoring the performance of two anaerobic digesters. These two digesters were fed with lipase treated and untreated restaurant wastewater that was neutralised with KOH solution prior to feeding. The oil-floating problem was minimised by this saponification of fatty acids with potassium hydroxide. However, there was no clear sign of an improvement for the treatment efficiency of the anaerobic digesters in terms of COD removal and methane production rate resulted in digesting lipase treated oily wastewater when compared to the one without lipase pre-treatment.

<p>The major part of this thesis describes the synthesis of enantiopure alcohols and diols by combining ruthenium-catalyzed redox reactions that lead to racemization or epimerization and lipase-catalyzed asymmetric trans-formations in one-pot.</p><p>A mechanistic study of the unexpected facile formation of <i>meso</i>-diacetate products found in enzyme-catalyzed acetylations of alkanediols with <i>Candida antarctica</i> lipase B (CALB) was first performed. By deuterium labeling it was found that the formation of <i>meso</i>-diacetates proceeds via different mechanisms for 2,4-pentanediol and 2,5-hexanediol. Whereas the first reacts via an intramolecular acyl migration, the latter proceeds via a direct, anomalous S-acylation of the alcohol. The acyl migration occurring in the 2,4-pentanediol monoacetate was taken advantage of in asymmetric transformations of substituted 1,3-diols by combining it with a ruthenium-catalyzed epimerization and an enzymatic transesterification using CALB. The in situ coupling of these three processes results in de-epimerization and deracemization of acyclic, unsymmetrical 1,3-diols and constitutes a novel dynamic kinetic asymmetric transformation (DYKAT) concept.</p><p>Racemization of secondary alcohols effected by a new ruthenium complex was combined in one-pot with an enzyme-catalyzed transesterification, leading to a chemoenzymatic dynamic kinetic resolution (DKR) operating at room temperature. Aromatic, aliphatic, heterocyclic and functionalized alcohols were subjected to the procedure. A mechanism for racemization by this ruthenium complex has been proposed and experimental indications for hydrogen transfer within the coordination sphere of ruthenium were found. The same ruthenium catalyst was used for epimerization in DYKAT of 1,2-diols, and a very similar complex was employed in isomerization of allylic alcohols to saturated ketones. The former method is a substrate extension of the above principle applied for DYKAT of 1,3-diols. The combination of a lipase and an organocatalyst was demonstrated by linking a lipase-catalyzed transesterification to a proline-mediated aldol reaction for the production of enantiopure (<i>S</i>)-<i>β</i>-hydroxy ketones and acetylated (<i>R</i>)-aldols.</p>

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mso-tstyle-colband-size:0 / mso-style-noshow:yes / mso-style-priority:99 / mso-style-parent:"" / mso-padding-alt:0cm 5.4pt 0cm 5.4pt / mso-para-margin-top:0cm / mso-para-margin-right:0cm / mso-para-margin-bottom:10.0pt / mso-para-margin-left:0cm / line-height:115% / mso-pagination:widow-orphan / font-size:11.0pt / font-family:"Calibri","sans-serif" / mso-ascii-font-family:Calibri / mso-ascii-theme-font:minor-latin / mso-hansi-font-family:Calibri / mso-hansi-theme-font:minor-latin / mso-bidi-font-family:"Times New Roman" / mso-bidi-theme-font:minor-bidi / mso-fareast-language:EN-US / } </style> <![endif]--> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (&lt / C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (&gt / C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to the</span><span style="font-size: 10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / mso-fareast-font-family:ArialOOEnc">&alpha / /&beta / hydrolase fold-3 domain protein of </span><i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Burkholderia </span></i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">sp. The RHgene34 protein </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into <i>Escherichia coli </i>Rosetta. <i>p</i>-Nitrophenol (<i>p</i>-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Kinetic analysis revealed that RHgene34 had a broad range activity on the <i>p</i>-Npesters, from C2 - C14. RHgene34 operates optimally at 45 &ordm / C, pH 9.0 and has a half life of 30 mins at 45 &ordm / C. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome.</span></p> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br /> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br />

The major part of this thesis describes the synthesis of enantiopure alcohols and diols by combining ruthenium-catalyzed redox reactions that lead to racemization or epimerization and lipase-catalyzed asymmetric trans-formations in one-pot. A mechanistic study of the unexpected facile formation of meso-diacetate products found in enzyme-catalyzed acetylations of alkanediols with Candida antarctica lipase B (CALB) was first performed. By deuterium labeling it was found that the formation of meso-diacetates proceeds via different mechanisms for 2,4-pentanediol and 2,5-hexanediol. Whereas the first reacts via an intramolecular acyl migration, the latter proceeds via a direct, anomalous S-acylation of the alcohol. The acyl migration occurring in the 2,4-pentanediol monoacetate was taken advantage of in asymmetric transformations of substituted 1,3-diols by combining it with a ruthenium-catalyzed epimerization and an enzymatic transesterification using CALB. The in situ coupling of these three processes results in de-epimerization and deracemization of acyclic, unsymmetrical 1,3-diols and constitutes a novel dynamic kinetic asymmetric transformation (DYKAT) concept. Racemization of secondary alcohols effected by a new ruthenium complex was combined in one-pot with an enzyme-catalyzed transesterification, leading to a chemoenzymatic dynamic kinetic resolution (DKR) operating at room temperature. Aromatic, aliphatic, heterocyclic and functionalized alcohols were subjected to the procedure. A mechanism for racemization by this ruthenium complex has been proposed and experimental indications for hydrogen transfer within the coordination sphere of ruthenium were found. The same ruthenium catalyst was used for epimerization in DYKAT of 1,2-diols, and a very similar complex was employed in isomerization of allylic alcohols to saturated ketones. The former method is a substrate extension of the above principle applied for DYKAT of 1,3-diols. The combination of a lipase and an organocatalyst was demonstrated by linking a lipase-catalyzed transesterification to a proline-mediated aldol reaction for the production of enantiopure (S)-β-hydroxy ketones and acetylated (R)-aldols.

A triglyceride lipase gene FgLip1 was identified in the genome of <i>Fusarium graminearum</i> strain PH-1. Yeast cells overexpressing FgLip1 showed lipolytic activity against a broad range of triglyceride substrates. Northern blot analyses revealed that expression of FgLip1 was activated in planta during the fungal infection process and under starvation conditions <i>in vitro</i>. FgLip1 expression was strongly induced in minimal medium supplemented with wheat germ oil, but only weakly induced by olive oil and triolein. Saturated fatty acids were the strongest inducers for FgLip1 expression and this induction was proportionally suppressed by the presence of unsaturated fatty acids. To determine the potential function of FgLip1, gene replacement was conducted on strain PH-1. When compared to wild-type PH-1, ∆FgLip1 mutants showed greatly reduced lipolytic activities at the early stage of incubation on minimal medium supplemented with either saturated or unsaturated lipid as the substrate, indicating that FgLip1 encodes a secreted lipase for exogenous lipid hydrolysis. The ∆FgLip1 mutants exhibited growth deficiency on both liquid and solid minimal media supplemented with the saturated triglyceride tristearin as the sole carbon source, suggesting that FgLip1 is required for utilization of this substance. No variation in disease symptoms between the ∆FgLip1 mutants and the wild-type strain was observed on susceptible cereal hosts including wheat, barley and corn. To delineate the promoter region responsible for the specific regulation of FgLip1 expression, a series of deletions of FgLip1 5 upstream region were fused with the open reading frame of a green florescent protein (GFP) gene and the constructs were introduced into <i>F. graminearum</i>. GFP expression in the resulting transformants indicated that a 563-bp FgLip1 promoter sequence was sufficient to regulate expression of the FgLip1 gene and regulatory elements responsible for gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements within the 563-372 bp region were mutated using a linker-scanning mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be responsible for FgLip1 basal expression, glucose suppression and fatty acid induction, respectively.

A triglyceride lipase gene FgLip1 was identified in the genome of <i>Fusarium graminearum</i> strain PH-1. Yeast cells overexpressing FgLip1 showed lipolytic activity against a broad range of triglyceride substrates. Northern blot analyses revealed that expression of FgLip1 was activated in planta during the fungal infection process and under starvation conditions <i>in vitro</i>. FgLip1 expression was strongly induced in minimal medium supplemented with wheat germ oil, but only weakly induced by olive oil and triolein. Saturated fatty acids were the strongest inducers for FgLip1 expression and this induction was proportionally suppressed by the presence of unsaturated fatty acids. To determine the potential function of FgLip1, gene replacement was conducted on strain PH-1. When compared to wild-type PH-1, ∆FgLip1 mutants showed greatly reduced lipolytic activities at the early stage of incubation on minimal medium supplemented with either saturated or unsaturated lipid as the substrate, indicating that FgLip1 encodes a secreted lipase for exogenous lipid hydrolysis. The ∆FgLip1 mutants exhibited growth deficiency on both liquid and solid minimal media supplemented with the saturated triglyceride tristearin as the sole carbon source, suggesting that FgLip1 is required for utilization of this substance. No variation in disease symptoms between the ∆FgLip1 mutants and the wild-type strain was observed on susceptible cereal hosts including wheat, barley and corn. To delineate the promoter region responsible for the specific regulation of FgLip1 expression, a series of deletions of FgLip1 5 upstream region were fused with the open reading frame of a green florescent protein (GFP) gene and the constructs were introduced into <i>F. graminearum</i>. GFP expression in the resulting transformants indicated that a 563-bp FgLip1 promoter sequence was sufficient to regulate expression of the FgLip1 gene and regulatory elements responsible for gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements within the 563-372 bp region were mutated using a linker-scanning mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be responsible for FgLip1 basal expression, glucose suppression and fatty acid induction, respectively.

Serum lipase activity has traditionally been used for diagnosis of pancreatitis in human beings and dogs. However, serum lipase activity is not specific for exocrine pancreatic function and many cell types other than pancreatic acinar cells also synthesize lipases. Recently, an immunoassay for the measurement of canine pancreatic lipase immunoreactivity has been developed and validated. This assay has shown to be specific for exocrine pancreatic function and sensitive for the diagnosis of canine pancreatitis. The objectives of this project were to purify feline pancreatic lipase (fPL), have antibodies against fPL (anti-fPL antibodies) produced, and develop a radioimmunoassay (RIA) for the diagnosis of feline pancreatitis. Pancreatic lipase was purified from feline pancreatic tissue by delipidation, anion-exchange chromatography, size-exclusion chromatography, and cation-exchange chromatography. Antiserum against fPL was raised in rabbits. Tracer was produced by iodination (125I) of fPL using the chloramine T method. An RIA was established and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intraassay variability, and inter-assay variability. A control range for fPLI in cat serum was established from 30 clinically healthy cats using the central 95th percentile. The sensitivity of the fPLI assay was 1.2 &#956;g/L. Observed to expected ratios for serial dilutions ranged from 58.0 to 164.3% for 4 different serum samples at dilutions of 1 in 2, 1 in 4, and 1 in 8. Observed to expected ratios for spiking recovery ranged from 76.0 to 156.5% for 4 different serum samples and 6 different spiking concentrations. Coefficients of variation for intra-assay variability for 4 different serum samples were 10.1, 4.5, 2.2, and 3.9%. Coefficients of variation for inter-assay variability for 4 different serum samples were 24.4, 15.8, 16.6, and 21.3%. The control range for serum fPLI concentration was established as 1.2 to 3.8 &#956;g/L. All of the objectives outlined above were successfully met, leading to the development of an RIA for the measurement of fPLI in cat serum. The RIA for fPLI described here is sufficiently accurate and precise, but has a limited linearity and reproducibility in the lower and higher end of the working range.

The present study cloned successfully two lipid-metabolism genes, lipoprotein lipase (LPL) and fatty acid binding protein (FABPs) from cobia and studied the mRNA expressions of the two genes and their upstream gene PPARs when the cobia were fed diets containing 15% lipid. Among the lipids, 6% was fish oil and the remaining 9% were supplemented by fish oil (FO, rich in n-3 HUFA), perilla oil (PE, rich in 18:2 n-6), safflower oil (SA, rich in 18:2 n-6), olive oil (OL, rich in 18:1 n-9) or palm oil (PA, rich in 16:0). The whole sequences of LPL, liver-FABP (L-FABP) and muscle-FABP (M-FABP) encode 520, 126 and 133 amino acids, respectively. RT-PCR and real time PCR analyses based on these gene sequences show that the mRNA expressions of L-FABP and M-FABP in the tissue of the cobia were diet-specific. The mRNA expression of LPL, on the other hand, did not respond to the treatments, except in visceral fat depot. Linear regression analysis shows that the mRNA expression of LPL in the liver and muscle was positively (P<0.05) related to dietary fatty acids and ther concentration, but that in the visceral fat depot was negatively related. The mRNA expression of FABPs was also positively correlated with dietary fatty acid levels. Among all fatty acids, the levels of C14:0, C20:1 n-9, EPA and DHA were positively correlated with the mRNA expression of PPAR£^and also with FABPs mRNA expression in the visceral fat depot and LPL mRNA expression in the muscle. Thus, LPL, L-FABP and M-FABP mRNA expression of the cobia were highly influenced by the kind and amount of dietary fatty acids. The role of PPARs was not clearly demonstrated.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 82-89).

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 173-189). Also available on the World Wide Web.