Dissertations / Theses on the topic 'Enzymes Separation'

The three main components of wood, namely, cellulose, hemicellulose, and lignin, can be used in various areas. However, since lignin covalently crosslinks with wood polysaccharides creating networks that is an obstacle for extraction, direct extraction of different wood components in high yield is not an easy matter. One potential approach to overcome such obstacles is to treat the wood with specific enzymes that degrade the networks by specific catalysis. However, the structure of wood is so compact that the penetration of the wood fibers by large enzyme molecules is hindered. Thus, the pretreatment of wood prior to the application of enzymes is necessary, for “opening” the structure. One pretreatment method that was performed in this thesis is based on kraft pulping, which is a well-established and industrialized technique. For untreated wood, the wood fibers cannot be attacked by the enzymes. A relatively mild pretreatment was sufficient for wood polysaccharides hydrolyzed by a culture filtrate. A methanol-alkali mixture extraction was subsequently applied to the samples that were pretreated with two types of hemicellulases, Gamanase and Pulpzyme HC, respectively. The extraction yield increased after enzymatic treatment, and the polymers that were extracted from monocomponent enzyme-treated wood had a higher degree of polymerization. Experiments with in vitro prepared lignin polysaccharide networks suggested that the increased extraction was due to the enzymatic untying. However, the relatively large loss of hemicellulose, particularly including (galacto)glucomannan (GGM), represents a problem with this technique. To improve the carbohydrate yield, sodium borohydride (NaBH4), polysulfide and anthraquinone were used, which increased the yields from 76.6% to 89.6%, 81.3% and 80.0%, respectively, after extended impregnation (EI). The additives also increased the extraction yield from approximately 9 to 12% w/w wood. Gamanase treatment prior to the extraction increased the extraction yield to 14% w/w wood. Sodium dithionite (Na2S2O4) is an alternative reducing agent for the preservation of hemicelluloses because it is less expensive than metal hydrides and only contains sodium and sulfur, which will not introduce new elements to the recovery system. Moreover, Na2S2O4has the potential to be generated from black liquor. Na2S2O4 has some preservation effect on hemicelluloses, and the presence of Na2S2O4 also contributed to delignification. The extraction yield increased to approximately 15% w/w wood. Furthermore, Na2S2O4 has been applied in the kraft pulping process of spruce. The yield and viscosity increased, while the Klason lignin content and kappa number decreased, which represents a beneficial characteristic for kraft pulp. The brightness and tensile strength of the resulting sheets also improved. However, the direct addition of Na2S2O4 to white liquor led to greater reject content. This problem was solved by pre-impregnation with Na2S2O4 and/or mild steam explosion (STEX) prior to the kraft pulping process. Following Na2S2O4 pre-impregnation and mild STEX, the obtained kraft pulp had substantially better properties compared with the properties exhibited after direct addition of Na2S2O4 to the white liquor. The wood structure opening efficiency of mild STEX alone was also tested. The accessibility of the wood structure to enzymes was obtained even at very modest STEX conditions, according to a reducing sugar analysis, and was not observed in untreated wood chips, which were used as a reference. The mechanical effect of STEX appears to be of great importance at lower temperatures, and both chemical and mechanical effects occur at higher STEX temperatures.

QC 20140903

Orientador: Elias Basile Tambourgi
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
Made available in DSpace on 2018-07-28T22:32:01Z (GMT). No. of bitstreams: 1 Rodrigues_ElianaMariaGoncalves_D.pdf: 3569293 bytes, checksum: b7c2f26dc727eabf16b3528d0a1813bb (MD5) Previous issue date: 2001
Resumo: Neste trabalho, foi estudada uma microcoluna agitada por campânulas pulsadas, visando promover um eficiente contato entre as fases através de uma agitação suave, aumentando assim o tempo de contato entre elas no interior da microcoluna e também evitando a desnaturação da enzima. O objetivo deste estudo visou a recuperação da xilanase, produzida pelo fungo Penicillium janthinellum, através da técnica de extração líquido - líquido por micela reversa, para o interior micelar. Para tanto, foi utilizado o agente tensoativo catiônico BDBAC (c1oreto de benzil dodecil bis (hidroxietil) amônio). Foram utilizados planejamentos estatísticos com o intuito de se realizar uma triagem das variáveis significativas no processo: freqüência de pulsação das campânulas, razão entre as fases aquosa/orgânica e condutividade da fase aquosa; a metodologia de superficie de resposta foi empregada para a quantificação dos níveis das mesmas. O trabalho resultou em dois modelos matemáticos, um que representa a recuperação da enzima pura, e outro que representa a recuperação da enzima presente no extrato enzimático bruto. Foram previstos e observados experimentalmente para ambos os modelos obtidos, rendimento em atividade na ordem de 140% para a enzima pura e 43% para o extrato enzimático. Verificou-se neste trabalho que, a metodologia estatística empregada foi de extrema importância e que, a microcoluna estudada tem operação estável e altos rendimentos em atividade foram alcançados
Abstract: In this work, it was studied a microcolumn agitated by puIsed caps, due to promote an efficient contact between the phases in the column and also to avoid the enzyme denaturation and the loss of main proteins properties. This work deals with the purification of xylanase produced by Penicillium janthinellum by liquid-liquid extraction on reverse micelles, to micellar inner, utilizing a cationic surfactant BDBAC (N-benzyI-N-dodecyl-N-bis(2-hydroxyethyl)). It was used design statistical with the intention of realized selection of the main variables in the process: frequency pulsed, volumetric flow and ionic strength; the response surface methodology was employed to the quantified levels of this. This resulted in two major mathematical models: one representing the use of pure enzyme and the other the use of enzymatic extract. It was predict and observed experimentally for both models, obtained activity yield in the order of 140% to the pure enzyme and 43% to the enzymatic extract. It was observed in this work that, the methodology statistical employed was extremely important and that the microcolumn used were stable operation and that high activity yield was reached.
Doutorado
Desenvolvimento de Processos Biotecnologicos
Doutor em Engenharia Química

La structure et le régulation des enzymes de synthèse des catécholamines ont été étudiées dans le phéochromocytome de rat. Nous avons mis au point un protocole permettant la séparation des trois premiers enzymes de la voie de synthèse. Ceci a permis de purifier partiellement la dopamine beta -hydroxylase et de mettre en évidence son inhibiteur endogène, qui a été décrit pour la première fois. Nous avons montré qu'une activité protéine kinase était copurifiée avec la tyrosine hydroxylase, qu'elle phosphoryle. Cette activité a été caractérisé et la séquence primaire du site de phosphorylation a été déterminée.

The aim of this work was to investigate the problems involved in production and separation of a yeast microsomal enzyme, cytochrome P450. Immobilisation of the microsomal preparation, and utilisation of the immobilised system for the removal of polycyclic aromatic hydrocarbons, particularly the carcinogen benzo(a)pyrene [B(a)P], from aqueous systems was also investigated. High recoveries of the microsomal enzyme were obtained using rapid, low-speed centrifugation (5 minutes, 3000xg) by prior precipitation with a non-ionic polymer, polyethylene glycol (PEG). PEG was found to effectively replace centrifugal acceleration. A semi-empirical mathematical model of the process based on the size distribution of the agglomerates formed and the proportion of the agglomerates sedimented by centrfugation was developed. The effect of PEG was consistent with its increasing the mean effective diameter of protein agglomerates in proportion to PEG concentration to an exponent of 0.619 and with its increasing the spread of the size distribution in proportion to the mean effective agglomerate diameter. Binding spectra studies established that B(a)P binds to the active site of yeast cytochrome P450 and is unaffected by PEG. The B(a)P assay by fluorescence following hexane extraction was also unaffected by PEG. The stability of the microsomal cytochrome P450 preparation at different temperatures was investigated. The enzyme half-life was 66 minutes at 37° and was also unaffected by PEG. The enzyme preparation was satisfactorily immoblilsed by encapsulation in calcium alginate gel and enzyme distribution profiles were determined in sections of alginate stained with coomasie blue by a novel technique using a scanning optical densitometer, Diffusivity coefficients of B(a)P and NADP in alginate gel beads were determined as 7.5 and 2.5 x 10[-10] m[2]/S, respectively by the Tanaka method, fitting solute depletion profiles to Crank's theoretical model. The microsomal enzyme preparation immobilised in alginate beads was used to remove B(a)P from aqueous solution. B(a)P removal was shown to be by a non-specific affinity absorption mechanism and the removal profile was found to correspond well to a theoretical model of a diffusion-reaction system, for an affinity ligand system. The activaiton energy for deactivation of microsomal P450 was measured as 33.56kJ/mole.

In this study, a methodology for separation of chiral molecules, by using enhanced ultrafiltration system was developed. Benzoin was the model chiral molecule studied. In the scope of developing this methodology, some parameters were investigated in the preliminary ultrafiltration experiments in order to set the operation conditions for enhanced ultrafiltration experiments. Due to the slight solubility of benzoin in pure water, 15% (v/v) Polyethylene glycol (PEG 400) and 30 % (v/v) Dimethyl sulfoxide (DMSO) were selected as cosolvents. Because of the high retention capacity of RC-10000 Da membranes for benzoin, a membrane saturation strategy was developed. In polymer enhanced ultrafiltration (PEUF) experiments bovine serum albumin (BSA) was used as ligand. Effects of ligand concentration and pH on total benzoin retention and on enantiomeric excess (ee %) were investigated. Benzoin concentration was almost kept constant at ~10 ppm and ~50 ppm for 15% (v/v) PEG 400 and 30 % (v/v) DMSO cosolvents, respectively. It was observed that the increase either in pH or in BSA concentration yielded an increase in total benzoin retention. In 15% (v/v) PEG 400-water, with BSA concentration of 10000 ppm, at pH 10, total benzoin retention reached to 48.7%. For this cosolvent, at different pH values and at different BSA concentrations, all ee % values were about or less than 10%. When 50000 ppm BSA was dissolved in 30 % (v/v) DMSO-water, total benzoin retention increased to 41.3% at pH 10 and ee % reached 16.7 % at pH 11. In enzyme enhanced ultrafiltration (EEUF) experiments, specific to benzoin, apo form of Benzaldehyde Lyase (BAL, E.C. 4.1.2.38) was used as ligand. These experiments were performed with constant ~ 10 ppm benzoin concentration in only 15% (v/v) PEG 400 &ndash
water solvent. Effect of BAL concentration on total benzoin retention and ee% was investigated. It was found that
for all the studied BAL concentrations in the range of 650- 1936 ppm total benzoin retention and ee % were kept almost constant at ~75% and ~60%, respectively.

The isotope partitioning studies of the Ascaris suum NAD-malic enzyme reaction were examined with five transitory complexes including E:NAD, E:NAD:Mg, E:malate, E:Mg:malate, and E:NAD:malate. Three productive complexes, E:NAD, E:NAD:Mg, and E:Mg:malate, were obtained, suggesting a steady-state random mechanism. Data for trapping with E:14C-NAD indicate a rapid equilibrium addition of Mg2+ prior to the addition of malate. Trapping with 14C-malate could only be obtained from the E:Mg2+:14C-malate complex, while no trapping from E:14C-malate was obtained under feasible experimental conditions. Most likely, E:malate is non-productive, as has been suggested from the kinetic analysis. The experiment with E:NAD:malate could not be carried out due to the turnover of trace amounts of malate dehydrogenase in the pulse solution. The equations for the isotope partitioning studies varying two substrates in the chase solution in an ordered terreactant reaction were derived, allowing a determination of the relative rates of substrate dissociation to the catalytic reaction for each of the productive transitory complexes. NAD and malate are released from the central complex at an identical rate, equal to the catalytic rate.

Automated and continuous processes are the future trends in downstream protein purification. A functionalized nanometer-scale membrane electrode system, mimicking the function of cell wall transporters, can selectively capture genetically modified proteins and subsequently pump them through the system under programmed voltage pulses. Numerical study of the two-step pulse pumping cycles coupled with experimental His-GFP releasing study reveals the optimal 14s/1s pumping/repel pulse pumping condition at 10 mM bulk imidazole concentration in the permeate side. A separation factor for GFP: BSA of 9.7 was achieved with observed GFP electrophoretic mobility of 3.1×10-6 cm2 s-1 V-1 at 10 mM bulk imidazole concentration and 14 s/1 s pumping/repel duration. The purification of His6-OleD Loki variant directly from crude E. coli extracts expression broth was demonstrated using the pulse pumping process, simplifying the separation process as well as reducing biopharmaceutical production costs. The enzymatic reactions showed that His6-OleD Loki was still active after purification. A nanoporous membrane/electrode system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzymes-complex system was demonstrated. The substrates residence time on the immobilized enzyme can be precisely controlled by changing the pumping rate and thereby prevent a secondary hydrolysis reaction. Immobilized enzyme showed long term storage longevity with activity half-life of 50 days at 4℃ and the ability to be regenerated. One-step immobilization and purification of His-tagged OleD Loki variant directly from expression broth, yielded 98% Uridine Diphosphate glycosylation and 80% 4-methylumbelliferone glycosylation conversion efficiency for the sequential reaction. A flow-through electroporation system, based on a novel membrane/electrode design, for the delivery of membrane-impermeant molecules into Model Leukocyte cells was demonstrated. The ability to apply low voltage between two short distance electrodes contributes to high cell viability. The flow-through system can be easily scaled-up by varying the micro-fluidic channel geometry and/or the applied voltage pulse frequency. More importantly, the system allows the electrophoretical pumping of molecules from the reservoir across the membrane/electrode system to the micro-fluidic channel for transfection, which reduces large amount of reagents used.

Différents travaux de recherche ont été décrits dans cette thèse. Les travaux de recherche peuvent être résumés comme suit. Le premier chapitre a porté sur l'identification d’inhibiteurs puissants efficaces vis-à-vis de de l'isoenzyme anhydrase carbonique humaine I (hCAI). Considérant l'importance pharmacologique de trouver des inhibiteurs (CAIs) et des activateurs (AACs) sélectifs aux isoformes de l’anhydrase carbonique ), l'anhydrase carbonique humaine I (hCAI) a été confrontée en parallèle à diverses bibliothèques dynamiques constitutionnelles (CDL). Dans le deuxième chapitre, des réseaux constitutionnels dynamiques ont été préparés sous forme de systèmes membranaires liquides et solides agissant comme un réseau pour le transport spécifique des ions lanthanides. Le transport est basé sur la capacité de complexation des lanthanides (La + 3, Lu + 3, Eu + 3) avec les groupes polyéther fonctionnels situés dans les matériaux membranaires. Dans le troisième chapitre, l'approche proposée consiste en l'utilisation de membranes liquides ioniques supportées (SILMs) comprenant deux enzymes différentes de l'anhydrase carbonique, l’enzyme thermo-résistante SspCA et l'enzyme bovine-CA, qui catalysent la réaction de conversion réversible du CO2 en bicarbonate en favorisant la force motrice vers le transport de CO2. La stabilité des membrane, leur perméabilité vis-à-vis de CO2 et de N2 ainsi que la sélectivité idéale (CO2 / N2) ont été déterminées pour les membranes développées. Le quatrième chapitre porte sur la synthèse et la caractérisation de membranes polymères denses pour une application en séparation de gaz. Les mesures de perméabilité aux gaz des membranes polymères synthétisées ont montré que la perméabilité de CO2 est supérieure à celle des autres gaz testés (CH4 et N2). Dans le dernier chapitre, des membranes de PVDF ont été fonctionnalisées avec une enzyme, la phosphotriestérase (PTE), selon deux méthodes différentes pour construire un réacteur à membrane biocatalytique (BMR) avec pour finalité la bioconversion et la séparation sélective du substrat paraoxon. La première méthode met en œuvre une dispersion réversible de nanoparticules magnétiques de PTE qui est immobilisée à la surface de la membrane de PVDF sous l’effet d'un champ magnétique externe. A l’inverse, la seconde méthode porte sur le greffage chimique de l'enzyme PTE, après modification de la surface de la membrane de PVDF native (DAMP-GA-enzymatique). Les deux techniques d'immobilisation d'enzymes ont montré une bonne efficacité et une sensibilité à l'égard de la bioconversion du paraoxon dans les différentes conditions appliquées dans un réacteur à membrane biocatalytique (BMR).De façon globale, les concepts développés dans ce travail de thèse permettront d’ouvrir de nouvelles pistes de recherche allant vers le développement d'une membrane polymère sélective au transport d’ions, de gaz mais aussi active dans les réactions catalytiques enzymatiques grâce à un contrôle bio-moléculaire au niveau des matériaux membranaires
Different research works have been described in this thesis. The research works can be summarized as the following. The first chapter deals with the identification of effective potent inhibitors for the human carbonic anhydrase I (hCAI) isozyme. Considering the pharmacological importance to find selective CA inhibitors (CAIs) and CA activators (CAAs), human carbonic anhydrase I (hCAI) has been subjected to a parallel screening of various constitutional dynamic libraries (CDL). In the second chapter, constitutional dynamic networks have been used in liquid and solid membrane systems as a carrier network for transporting lanthanides. The transport is based on the complexing ability of lanthanides metals (La+3, Lu+3, and Eu+3) with the functional polyether groups in the membrane materials. In the third chapter, the proposed approach consists in using supported ionic liquid membranes (SILMs) comprising two different carbonic anhydrase enzymes, the thermo-resistant SspCA enzyme and the Bovine-CA enzyme, which catalyze the reaction of reversible conversion of CO2 to bicarbonate, enhancing the driving force for CO2 transport. Membrane stability, CO2 and N2 permeability and (CO2/N2) ideal selectivity were determined for the membranes developed. In the fourth chapter, the research work consists in the synthesis and characterization of dense polymeric membranes for gas separation application. The gas permeability measurements for the synthesized polymeric membranes showed that the permeability of CO2 is higher than other used gases (N2 and CH4). In the last chapter, two different methods of PVDF membrane functionalization with a phosphotriesterase (PTE) enzyme have been developed to construct biocatalytic membrane reactor (BMR) for bioconversion and selective separation of paraoxon substrate. The first method employs reversible dispersion of magnetic nanoparticle immobilized with PTE using an external magnetic field on the surface of native PVDF membrane. On the contrary, the second method comprises chemical grafting of the PTE enzyme, after surface modification of the native PVDF membrane (DAMP-GA-Enzyme). Both methods of enzyme immobilization showed good efficiency and sensitivity towards the bioconversion of paraoxon substrate at different conditions applied in a biocatalytic membrane reactor (BMR).In general, the concepts developed in this thesis research work will help bring new tracks on the way to the development of a polymeric membrane for selective ion and gas separation but also for selective catalytic reaction under bio(molecular) control

In this study, resolution of racemic mandelic acid by enyzme enhanced ultrafiltration (EEUF) was studied. In order to develop a methodology, bovine serum albumin (BSA) was used as a model protein for polymer enhanced ultrafiltration (PEUF) experiments and the enzyme S-mandelate dehydrogenase was used for EEUF experiments. To be used for enzyme enhanced ultrafiltration experiments, the gene which is responsible from the production of S-mandelate dehydrogenase was isolated from Pseudomonas putida, expressed in Escherichia coli and the recombinant enzyme was produced. For PEUF experiments, effects of pH and ligand ratio were investigated. Total retention of mandelic acid increased with decrease in pH and total retention of mandelic acid reached to a maximum value of 74.4% at pH 4.3. For EEUF experiments, pH and ligand ratio effect on total retention, enantiomeric excess, enantioselectivity were investigated. Although apoenzyme was tried to be obtained by diafiltration and conversion was tried to be prevented, conversion occured especially at high pH values. To create the apoenzyme effect, three methods were studied. Enzyme conversion was prevented by sodium sulfite inhibition but enzyme did not retain mandelic acid. By oxygen saturation of enzyme, conversion was prevented, binding was achieved but enzyme showed no enantioselectivity. When the enzyme was diafiltrated at pH 10.0, total mandelic acid retention, enantiomeric excess and enantioselectivity reached to 77.2%, 38.9%, 2.27, respectively and the enzyme selectivity was reversed as R-selective.

Casein is the major protein fraction in milk, and its cross-linking has been a topic of scientific interest for many years. Enzymatic cross-linking has huge potential to modify relevant techno-functional properties of casein, whereas non-enzymatic cross-linking occurs naturally during the storage and processing of milk and dairy products. Two size separation techniques were applied for characterisation of these reactions: gel electrophoresis and size exclusion chromatography. This review summarises their separation principles and discusses the outcome of studies on cross-linked casein from the last ~20 years. Both methods, however, show limitations concerning separation range and are applied mainly under denaturing and reducing conditions. In contrast, field flow fractionation has a broad separation range and can be easily applied under native conditions. Although this method has become a powerful tool in polymer and nanoparticle analysis and was used in few studies on casein micelles, it has not yet been applied to investigate cross-linked casein. Finally, the principles and requirements for absolute molar mass determination are reviewed, which will be of increased interest in the future since suitable calibration substances for casein polymers are scarce.

Content: In this study, different acid proteases, which were produced from Aspergillus and Bacillus, were applied for wet-blue bating and their properties and bating effects were observed. The results showed that the acid protease produced Aspergillus had better bating effect and higher chromium tolerance than that of produced by Bacillus. Furthermore, how the acid protease influenced wet-blue microstructure was analyzed by SEM and Micro-CT. The enzymatic properties of acid protease was studied firstly.Zeta potential analysis showed that the isoelectric point (pI) of the protease was consistent with its pH value, which was at 3.0. By particle size analysis, it found that its particle size was 700 nm. In order to obtain the functional components, the molecular weight of the acidic protease was analyzed by Polyacrylamide gel Electrophoresis (SDS-PAGE). Different molecular weight components were obtained by separating the acidic protease with Tangential Flow Filtration (TFF) Technology. The characteristics of these components were determined such as enzyme types and their proportion. Afterwards, these different molecular weight components were used for wet-blue bating. The bating effluent was collected, and then, contents of Hydroxyproline (Hyp), Hyaluronic acid (HA), Desmosine (Des) and Chondroitin sulfate (CS) were analysed, which could be directly corresponding with the degradation of different proteins in wet-blue. Therefore, by characterizing and comparing the bating effect influenced with these different molecular weight components, the functional components of protease could be identified and further be separated and purified. Based on these results, this research is helpful to the development and study of the action of acid protease in the wet-blue bating process. Take-Away: Micro-CT as a new way to characterize the microstructure of leather; Identification and Separation of Effective Components of Acid Protease； Degradation Analysis of Main Components of Wet-Blue during bating process.

Currently, the transplantation of islets of Langerhans is a viable means to maintain control of blood sugar levels and reduce the risk of hypoglycemia in defined populations with brittle type I diabetes mellitus or those requiring pancreatectomy. However, the process of islet isolation is highly variable and not all isolations result in islet numbers or quality suitable for transplantation. This thesis aimed to improve transplantation success through optimization and standardization of the isolation process and to identify pretransplant variables associated with early islet engraftment. A previously disregarded enzyme activity, tryptic-like activity (TLA), has been identified to influence pancreas digestion efficiency and islet isolation success in both the preclinical and clinical situations. For human pancreases, islet isolation success rates improved from 0% in the lowest TLA group to over 50% in the highest TLA groups without affecting islet quality. These findings should help standardize evaluation of enzymes for clinical islet isolation. A closed, automated, pump-made gradient system was compared to the open, manual method for islet separation. No differences were observed in expected gradient volumes, islet yields or total purities between the two methods. The pump-made gradient system successfully removed manual influences on density gradient production while fulfilling regulatory requirements for closed system processing. Islet quantification was evaluated with computer-assisted digital imaging analysis (DIA) and a semi-closed assessment system. By using the DIA system method, which measures islet purity and pellet volume instead of manual counting methods, variation in islet counts and purity reduced by almost half. By using a transplant outcome measurement of C-peptide adjusted by blood glucose and creatinine, we identified four pretransplant factors that affect early transplant outcome. Of the four factors, one was related to the organ transport time, one to function of the islets, and two to the transplanted tissue volume. When these four factors were put into a predictive model, it accounted for about 40% of the transplant outcome. The work contained in this thesis identifies and optimizes a number of critical elements related to islet isolation and transplantation protocols.

Processing sexual assault samples is a difficult time consuming task for the forensic analyst. Samples tend to be a mixture of the victim’s epithelial cells and the male suspect’s sperm cells that need to be separated prior to extraction of Deoxyribonucleic Acid (DNA). Without separation of the two cell types, the DNA extract would result in an uninterpretable mixture. In 1985, Peter Gill and colleagues outlined a procedure known as preferential lysis that would aid in the separation of female and male cells from sexual assault samples. The basis of this procedure, commonly referred to as a differential extraction, utilizes differences in the packaging of DNA between the two cell types to preferentially lyse the female epithelial cells and leave the sperm intact. By pelleting the sperm and removing the supernatant (termed the Non-Sperm Fraction) the Sperm Fraction can now be extracted without the contaminating epithelial cells. This procedure has been widely implemented in forensic laboratories and is still being used today over 30 years later. However, there are certain conditions under which this procedure does not perform sufficiently including excess of female epithelial cells and low amounts of sperm. Unfortunately, both of these conditions are common among sexual assault samples. The procedure also is quite long, and with the backlog of sexual assault samples continually growing in the United States, there is a need for a new procedure that is faster and performs optimally under the previously mentioned conditions. This research explores the use of two enzymes, EA1 (marketed by Zygem Corporation as ForensicGEM Saliva™) and Trypsin to separate the cells. Due to the inability of Zygem to cleave the disulfide bonds present in the sperm DNA packaging proteins, treatment of mixed samples with Zygem will lyse the epithelial cells and leave the sperm intact. The incubation time of Zygem is much faster than that of the Gill method and can be performed in one tube, minimizing the chances of DNA loss and contamination during transfers. Treatment of the pelleted Zygem extract with Trypsin effectively and rapidly lyses the sperm cells. Combining these methods into a differential extraction protocol has the potential to be a rapid, easily implemented procedure. Results from the Zygem-Trypsin differential extraction method showed incomplete separation of the two fractions due to the incomplete lysis of the epithelial cells by Zygem. The resultant profiles did show a major male contribution with a minor female component, however these results are not sufficient enough for real casework. While further research and development of the protocol are necessary, the Zygem-Trypsin differential extractions performed here show the potential for a rapid, easy differential extraction procedure that could be easily implemented in any laboratory.
2017-11-03T00:00:00Z

碩士
國立屏東科技大學
食品科學系
94
Commercial pectolytic enzymes play an important role in the winemaking process. PE (pectinesterase) can catalysis the de-esterification reaction、the methyl ester group on the C6 carboxyl of D- galacturonic acid in the pectin could be hydrolyzed by PE to produce the free carboxyl group and the methanol、and the methanol is harmful to human body. Affinity chromatography re-esterification CL-AIS can separate PL from commercial pectolytic enzymes、but it was hard to separate PG (polygalacturonase) from PE. In winemaking、Black queen grape was used as the raw material in this study、and it was fermented with commercial yeast RA-17 (S. cerevisiar). Commercial pectolytic enzymes、commercial pectolytic enzymes without PL and PL were added in grape winemaking individually、Lab value、pH value、%T、titrable acidity(%)、toatal soluble solids(°Brix)、methanol and alcohol content were detected during the procedure of winemaking. The methanol content in the sample added with commercial pectolytic enzymes was 3102ppm (based on ethanol content) and the methanol content was only 563ppm as the PL enzyme was added only. The result showed that the PL (pectin lyase) enzyme used in the winemaking could reduce the methanol content and the must was clear、so、the development of PL enzyme is promising for the clarification and reducing the content of the methanol in fruit juice and wine in the future.

PARP-1 is a nuclear enzyme involved in a range of activities associated with DNA metabolism, playing a key role in maintaining the integrity of DNA and chromatin structure. As such, this enzyme is likely to provide a useful target when using a rationale drug design approach to develop pharmaceutical reagents including cancer therapeutics. A major obstacle to this work however is that our knowledge of the relationship between structure and function of PARP-1 is rather limited. Structure - function studies of enzymes typically require the application of techniques for producing reasonable quantities of high quality protein. To this end, this thesis describes the development of a rapid and relatively simple approach to producing and purifying PARP-1.

博士
國立成功大學
化學工程學系碩博士班
93
This thesis is divided into two parts. The first part is aimed at the investigation of immobilized metal ion affinity adsorbent for a-amylase separation and the second part is on the development and validation of analysis methods, liquid chromatography and capillary electrophoresis, for a novel chemotherapeutic enhancer, methoxyamine. b-Cyclodextrin (b-CD) and epichlorohydrin (EPI) were chosen as the matrix material and crosslinker, respectively. Thus the cross-linked b-cyclodextrin (b-CDcl) could react with iminodiacetic acid (IDA) to form b-CDcl-IDA. The immobilized metal ion adsorbent, b-CDcl-IDA-Cu2+, was prepared by chelating metal ions with the carboxylic groups from IDA. The adsorbent could adsorb 750 U/mL Bacillus licheniformis a-amylase (BLA) within 2 min. However, it took 2 hours to reach the adsorption equilibrium as the BLA activity was raised to 38,900 U/mL. A rapid and repeated BLA adsorption-desorption procedure was executed 50 times with 500 mg of adsorbent and the average recovery of 97% was achieved. In total, 93% of recovery and 46-fold concentration could thus be obtained. 　The presence of PEG could facilitate a-amylase adsorption from Bacillus amyloliquefaciens a-amylase (BAA) fermentation broth. 95% of adsorption could be obtained by changing the molecular weight and the amount of PEG. Adding 200 mM NaCl to the desorption agent, imidazole, the desorption of BAA raised by 35% to more than 95%. The BAA were separated from the fermentation broths by the prepared adsorbents and the BAA recovery were higher than 80%. Esterase was used to examine the specific affinity of adsorbents for a-amylase. In the mixture of a-amylase/esterase, the adsorption of a-amylase was nearly not influenced by esterase. On the contrary, the esterase adsorption was inhibited by a-amylase. 　In the second part, UV-Visible photometer, LC-MS and chemical reduction were used to discover the derivatization reaction of derivating agent, 4-(diethylamino)benzaldehyde (DEAB), with methoxyamine. The high performance liquid chromatography (HPLC) method quantitated the main derivate, 4-(diethylamino)benzaldehyde o-methoxyloxime (DBMO), at the wavelength of 310 nm. A linear calibration range of 0.10-10.0 mM was obtained. Another method is using pH 2.5 phosphate buffer as the separation buffer for capillary electrophoresis (CE). The relative peak area of protonated 4-(diethylamino)benzaldehyde o-methoxyloxime (DBMOH+) to the internal standard, N,N-dimethyl-p-toluidine (DMPT), was adopted to the calibration which had a linear range of 5.0-500 mM at the wavelength of 200 nm.

碩士
國立成功大學
化學工程學系碩博士班
91
This thesis concerns the applications of iron oxide magnetic nanoparticles in enzyme immobilization and separation. In the former, Lipase was immobilized on Fe3O4 magnetic nanoparticles. The preparation conditions, product properties, and the performances in the water systems were investigated. In the latter, polyacrylic acid (PAA) was covalently bound onto Fe3O4 magnetic nanoparticles to be a novel nano-adsorbent. The preparation conditions, product properties, and the application in the adsorption of Bromelain in aqueous solution were investigated. Lipase was covalently bound onto Fe3O4 magnetic nanoparticles (12.7 nm) via carbodiimide activation. The Fe3O4 magnetic nanoparticles were prepared by co-precipitating Fe2+ and Fe3+ ions in an ammonia solution and treating under hydrothermal conditions. The analyses of transmission electron microscopy (TEM) and X-ray diffraction (XRD) showed that the size and structure of magnetic nanoparticles had no significant changes after enzyme binding. Magnetic measurement revealed the resultant lipase-bound magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu/g (only slightly lower than that of the naked ones (64 emu/g)), a remanent magnetization of 1.0 emu/g, and a coercivity of 7.5 Oe. The analysis of Fourier transform infrared (FTIR) spectroscopy confirmed the binding of lipase onto magnetic nanoparticles. Compared to the free enzyme, the bound lipase exhibited a 1.41-fold enhanced activity, a 31-fold improved stability, and better tolerance to the variation of solution pH . The kinetic behavior of bound Lipase was also determined in aqueous solution. A novel magnetic nano-adsorbent was prepared by covalently binding polyacrylic acid (PAA) on Fe3O4 magnetic nanoparticles (13.2 nm) via carbodiimide activation. From the analyses of TEM, XRD and magnetism, the magnetic nanoparticles showed no change in size, structure and superparamagnetic characteristics after binding PAA. The analyses of FTIR, thermogravimetric analysis (TGA), differential thermal analysis (DTA) and X-ray photoelectron spectroscopy (XPS) confirmed the binding of PAA to magnetic nanoparticles and suggested the binding mechanism of PAA. The ionic exchange capacity of the resultant magnetic nano-adsorbents was estimated to be 1.64 meq/g, much higher than those of the commercial ionic exchange resins. The full recovery of bromelain was achievable within 1 min onto the nano-adsorbents at pH 3-5 and 0.1 M phosphate when the concentration of bromelain was 6 mg/ml. Moreover, the complete desorption of bromelain from the nano-adsorbents was attained within 1 minute at pH 7 when the concentration of KCl was above 0.6 M. The isothermal adsorption indicated that the adsorption behavior of bromelain followed the Langmuir adsorption isothermal and the values of the maximum amount of adsorbed bromelain (qm) and Langmuir constant (K) were 0.476 mg/mg and 58.4 ml/mg, respectively. In addition, bromelain retained 87.4% activity after adsorption/desorption.

碩士
中原大學
電子工程學系
87
More recently the ion sensitive field effect transistor (ISFET) has been studied extensively because of some advantage, such as small size, rapid response and more importantly, the possibility of manufacturing by MOSFET processes. However, as far as we know, ISFET is limited by the structure for biological and chemical application. Therefore, it is important to study the structure of ISFET for useful application. In this thesis, our laboratory introduced a novel separative structure of EGFET. This structure is an improvement of extended gate structure ISFET. Tin oxide thin film was prepared by the sputtering system as a pH sensitive material which has been presented by our laboratory. The first topic is the first application of separative structure of EGFET with tin oxide thin film. The results show that separative structure of EGFET with tin oxide thin film has better characteristics for pH sensor. Another major topic in this thesis emphasized on the bio-application of the novel separative structure of EnFET. In this study, we firstly immobilized the enzyme on tin oxide pH-sensitive film, and investigated the effect of acetylcholinesterase immobilized processes to detect acetylcholine. We also discussed the parameters during the measurement for pesticide detection in the further.

Recently, there has been a growing drive towards the bottom-up development of synthetic cells that mimic key cellular features. A cellular feature ubiquitous amongst cells is that of compartmentalisation. Compartmentalisation enables the spatiotemporal control of biochemical reactions and is thus vital for the development of synthetic cells. To date, most synthetic cell models have utilised classical membrane bound containers as model compartments. However, recent advances in cell biology have highlighted the importance of membraneless compartments formed via liquid-liquid phase separation (LLPS) as organisation centres. It has been suggested that these organelles play a critical role in regulating cell biochemistry, yet very little is known about their interactions with enzymatic reactions. Thus, aiming to develop novel synthetic capabilities, the work presented in this thesis designs and characterises synthetic cells which include features of membraneless compartmentalisation. These systems utilise complex coacervates, a specific type of LLPS that is driven by the electrostatic attraction of oppositely charged polymers, as model membraneless compartments. These low complexity systems subsequently provide ideal platforms for systematic investigations of the interaction of membraneless coacervate compartments with enzymatic reactions. In Chapter 3 and 4, I focus on developing a responsive synthetic cell system that recapitulates features of membrane-bound and membraneless compartmentalisation. I generate a pH-responsive system by exploiting the intrinsic pKa of cationic polylysine to trigger coacervation within a liposome. This synthetic cell is then functionalised with the enzyme formate dehydrogenase (FDH). I show that coacervate properties can be utilized to locally concentrate and activate the FDH reaction at low enzyme concentrations, thus demonstrating that membraneless compartments can activate reactions via sequestration into coacervate reaction centres. In Chapter 5, I then proceed to characterise whether the diffusive exchange of molecules across a droplet phase boundary effects enzyme dynamics. Synthetic cells constructed from emulsion droplets with coacervate sub-compartments were used as model systems with diffusive exchange, while bulk coacervate and supernatant phases were used as uncoupled model systems without exchange. I studied the FDH reaction in both models and I conclude that coupling of the phases increases reaction rates compared to an uncoupled system. When coupled, the supernatant acts as a ’sink’ removing the product NADH from the coacervate droplets. This increases the apparent reaction rate in the supernatant, while the reduction of NADH concentration in the coacervate reduces product inhibition. This demonstrated that the open phase boundary tightly couples membraneless droplets to their surroundings, which can ultimately lead to increased reaction rates both inside and outside the compartments. Finally in Chapter 6, I scrutinize enzyme kinetics of the enzymes FDH and β -galactosidase in the unique coacervate physicochemical environment using Michaelis-Menten assays in CM-Dex/PDDA bulk phase. Results show that the KM and Vmax of FDH significantly increased compared to buffer, while those of β-galactosidase do not. I hypothesise that the negatively charged formate substrate of the FDH reaction interacts strongly with the positively charged PDDA, decreasing its affinity for the enzyme. Furthermore, I suggest that the coacervate environment facilitates the rate limiting hydride transfer of the reaction, thereby increasing the maximum rate. This data demonstrates that the coacervate environment itself can tune and control enzyme dynamics. In conclusion, my work establishes responsive, tunable and enzymatically active syn- thetic cellular systems with features of membraneless compartmentalisation. My results indicate that membraneless compartments can have significant impact on the dynamics of enzymatic reactions, opening up possible ways to control reaction rates in synthetic systems and suggesting plausible functions for membraneless organelles in vivo. Overall, I demonstrate that rationally designed synthetic cells provide biomimetic experimental platforms that offer insights into the influence of membraneless compartmentalisation on enzymatic reactions. Parts of the presented work have been published as two first author publications in peer-reviewed journals.
‘Bottom-up'’ Modelle synthetischer Zellen, die Schlüsselmerkmale zellbasierten Lebens imitieren, rücken immer mehr in den Fokus. Von zentraler Bedeutung ist hier die Kompartmentbildung. Sie erst ermöglicht die räumliche und zeitliche Kontrolle biochemischer Abläufe und ist daher entscheidend bei der Entwicklung synthetischer Zellen. Bisher wurden in der Mehrzahl der synthetischen Zellmodelle klassische, membrangebundene Reaktionsräume als Modellkompartimente verwendet. Jüngste Fortschritte in der Zellbiologie belegen jedoch die Bedeutung von membranlosen Kompartimenten, die durch Flüssig-Flüssig-Phasentrennung (LLPS) gebildet werden. Es wird angenommen, dass diese membranlosen Kompartimente eine zentrale Rolle bei der Regulierung der Zellchemie spielen. Jedoch ist bisher nur sehr wenig über ihren Einfluss auf enzymatische Reaktionen bekannt und experimentell belegt. Mit dem Ziel, die Bandbreite und das Verständnis synthetischer Modelle zu erweitern, wurden in dieser Arbeit neue Methoden entwickelt und dargestellt, die membranlose Kompartmentbildung benutzen. Es wurden hierfür komplexe Koazervate eingesetzt, eine spezielle Art der LLPS, welche durch die elektrostatische Anziehung von entgegengesetzt geladenen Polymeren angetrieben wird. Diese verhältnismäßig einfachen Systeme bieten eine ideale Plattform für systematische Untersuchungen des Einflusses von membranlosen Koazervatkompartimenten auf enzymatische Reaktionen. In den Kapiteln 3 und 4 konzentrierte ich mich auf die Entwicklung eines reaktionsfähigen synthetischen Modellsystems, das die Phänomene sowohl membrangebundener als auch membranfreier Kompartmentbildung vereint. Zur Steuerung der Koazervierung innerhalb von Liposomen wurde ein pH-reaktives System verwendet, welches sich den intrinsischen pKa von kationischen Polylysin zunutze macht. Diese synthetis- che Zelle wurde im folgenden Schritt mit dem Enzym Formiat-Dehydrogenase (FDH) funktionalisiert. Ich konnte damit zeigen, dass es die Eigenschaften von Koazervaten ermöglichen, die FDH-Reaktion bei global sehr niedrigen Enzymkonzentrationen zu aktivieren. Hierbei wirken die membranlosen Koazervate in Folge einer lokal er- höhten Enzymkonzentration als Zentren gesteigerter Reaktivität. Dies geschieht durch die lokale Konzentrationserhöhung in Koazervaten, was bei LLPS auch durch den Verteilungskoeffizient beschrieben wird. Mit anderen Worten agieren diese membran- losen Kompartimente durch Sequestrierung als Reaktionszentren. Im Kapitel 5 charakterisierte ich den Einfluss von diffusivem Molekülaustausch auf die Enzymkinetik über die Koazervat-Phasengrenze hinweg. Hierbei wurden zwei Systeme miteinander verglichen. Einerseits wurde ein synthetisches Zellmodell, beste- hend aus mikrofluidisch hergestellten Wasser-in-Öl Emulsionstropfen, die Koazervate enthalten, als Modellsystem mit diffusivem Austausch zwischen den Phasen verwendet. Andererseits wurden separate, reine Koazervatphasen und reine Überstandsphasen als Modellsysteme ohne Austausch verwendet. Ich habe die FDH-Reaktion in beiden Modellsystemen untersucht und kam zu dem Schluss, dass die Kopplung der Phasen die Reaktionsgeschwindigkeiten im Vergleich zu den ungekoppelten Systemen erhöht. Bei der Kopplung wirkt die Überstandsphase als Senke, die das Produkt NADH aus den Koazervaten aufnimmt. Dies erhöht die scheinbare Reaktionsgeschwindigkeit im Überstand, während die Verringerung der NADH-Konzentration im Koazervat die Produkthemmung verringert. Dies zeigt, dass die offene Phasengrenze membranloser Kompartimente eng mit ihrer Umgebung gekoppelt ist, was als erhöhte Reaktionsraten sowohl innerhalb als auch außerhalb der Kompartimente gemessen werden kann. Schließlich untersuchte ich in Kapitel 6 die Enzymkinetik der Enzyme FDH und β- Galaktosidase in der physikalisch-chemischen Umgebung des Koazervats. Mit Hilfe von Michaelis-Menten-Experimenten in der CM-Dextran/PDDA-Bulkphase konnte gezeigt werden, dass KM und Vmax von FDH im Vergleich zum Überstand signifikant erhöht sind, wohingegen jene von β-Galaktosidase ein solches Verhalten nicht zeigen. Das führte mich zu der Hypothese, dass das negativ geladene Formiatsubstrat der FDH- Reaktion stark mit dem positiv geladenen PDDA interagiert, wodurch seine Affinität für das Enzym abnimmt. Darüber hinaus wird der ratenbegrenzende Hydridtransfer in der Umgebung des Koazervats erleichtert und es kann eine Erhöhung der Reaktionsrate beobachtet werden. Die Daten zeigen, dass abhängig vom Koazervat-Milieu die Enzymdynamik in verschiedene Richtungen gesteuert werden kann. Zusammenfassend lässt sich sagen, dass meine Arbeit reaktionsfähige, steuerbare und enzymatisch aktive synthetische Zellsysteme mit Eigenschaften membranloser Kompartmentbildung etabliert. Meine Ergebnisse deuten darauf hin, dass membranlose Kompartimente einen signifikanten Einfluss auf die Dynamik enzymatischer Reaktio- nen haben. Meine Untersuchungen eröffnen damit neuartige Wege zur Kontrolle der Reaktionsgeschwindigkeit in synthetischen Systemen und erweitern das Verständnis möglicher Funktionen membranloser Organellen in vivo. Insgesamt zeige ich, dass über- legt entworfene synthetische Zellen eine hervorragende biomimetische Plattform bieten, um Einblicke in den Einfluss von membranloser Kompartimentierung auf enzymatische Reaktionen zu gewinnen. Teile der vorgestellten Arbeit wurden als wissenschaftliche Beiträge in zwei begutachteten Journalen als Erstautor veröffentlicht.