Dissertations / Theses on the topic 'Hen egg lysozymes'

Hen egg-white lysozyme (HEWL), a paradigmatic glycoside hydrolase (GH), is a retaining β-glycosidase catalysing the cleavage of β-glycosidic bonds found in the cell wall of Grampositive bacteria. Despite extensive research, the nature and role of substrate distortion in catalysis is still unclear. Here, MM and QM/MM modelling was performed on a HEWL trisaccharide (NAM-B-β(1→4)-NAG-C-β(1→4)-NAM-D) product complex to investigate distortion and reactivity of the NAM-D ring bound in the -1 site of the enzyme.

Deposition of protein fibers with a characteristic cross-β sheet structure is the molecular marker associated with human disorders ranging from Alzheimer's disease to type II diabetes and spongiform encephalopathy. Given the large number of non-disease related proteins and peptides that have been shown to form amyloid fibrils in vitro, it has been suggested that amyloid fibril formation represents a generic protein phase transition. In the last two decades it has become clear that the same protein/peptide can assemble into distinct morphologically and structurally amyloid aggregates depending on the solution conditions. Moreover, recent studies have shown that the early stage, oligomeric amyloid assemblies are the main culprit in vivo. We have investigated the amyloid assemblies formed under denaturing conditions for Hen Egg White Lysozyme (HewL) whose human homologue is directly implicated in hereditary non-neuropathic systemic amyloidosis. Our early investigations showed that HewL can aggregate via at least two distinct assembly pathways depending on solution ionic strength at fixed pH, temperature, and protein concentration. By combining Dynamic Light Scattering (DLS), Static Light Scattering (SLS) and Atomic Force Microscopy (AFM) we showed that at low ionic strength, the pathway is characterized by the nucleation and growth of long (several micron), rigid fibrils (RF) via monomers assembly. A second, high ionic strength pathway is characterized by the rapid assembly of monomers into globular oligomers that further polymerize into curvilinear fibrils (aO/CF). At NaCl concentrations above 400 mM, aggregation resulted in precipitate formation. Next, we used Foureir Transform Infrared spectroscopy (FTIR) and an amyloid-specific dye, Thioflavin T (ThT), to show that both RF and (a)O/CF are amyloidogenic species, but they have detectable structural differences. Moreover, we have determined that each assembly pathway has unique SLS, DLS, FTIR and ThT response signatures that help determine the assembly type prior to AFM imaging of aggregates. Taking advantage of the morphological, structural and kinetic signatures for the two distinct HewL amyloid aggregates I mapped out their amyloid aggregates phase diagram spanning over two orders of magnitude in protein concentration and from 50 to 800 mM NaCl in ionic strength. This is the most complete phase diagram for amyloid aggregates of a given protein up to date. The phase diagram has three distinct regions delineated by sharp boundaries. The RF- aO/CF was called Critical Oligomer Concentration, and we commonly refer to “above the COC” as the region were aO/CF are kinetically favored.. In the region of low salt/high protein concentrations, RF were the only amyloid species to nucleate and grow. As both salt and protein concentrations increase, aO/CF become the kinetically favored species, and RF nucleate and grow after several days of incubation. At high protein and high salt concentrations, aO/CF form very fast and eventually lose solubility forming a precipitate (Ppt). Cross-seeding experiments showed that RF is the thermodynamically stable aggregate phase, while the O/CF are the metastable species. Finally, we used the phase diagram to design experiments that would allow us to reveal the RF nucleation mechanism in presence of aO/CF. RF nucleation above the COC can undergo either via internal restructuring of aO/CF (NCC) or through a random coalescence of monomers into a nucleus (NP). The experimental results obtained so far strongly indicate that RF nucleate via NP mechanism both below and above the COC.

Advances in recent nanoscience technologies have generated a new compilation of biocompatible, fluorescent nanoparticles derived from semiconductor quantum dots (QDs). QDs are extremely small in size and possess very large surface areas, which gives them unique physical properties and applications distinct from those of bulk systems. When exposed to biological fluid, these QDs may become coated with proteins and other biomolecules given their dynamic nature. These protein-QD systems may affect or enhance the changes in protein structure and stability, leading to the destruction of biological function. It is believed that these QDs can act as nucleation centers and subsequently promote protein fibril formation. Protein fibrillation is closely associated with many fatal human diseases, including neurodegenerative diseases and a variety of systemic amyloidoses. This topic of protein-QD interaction brings about many key issues and concerns, especially with respect to the potential risks to human health and the environment. Herein, the behavioral effects of dihydrolipoic acid (DHLA)-capped CdSe/ZnS (core/shell) QDs in hen egg-white lysozyme (HEWL) and human serum albumin (HSA) protein systems were systematically analyzed. This study gives rise to a better understanding of the potentially useful application of these protein-QD systems in nanobiotechnology and nanomedicine as a bioimaging tool and/or as a reference for controlled biological self-assembly processes.

O aumento na procura por produtos naturais pelo mercado farmacêutico, cosmético e alimentício demanda pesquisas no desenvolvimento desses produtos. Esses são direcionados à obtenção de substâncias de origem vegetal ou animal, assim como, para produtos biotecnológicos. Investigações quanto à atividade antibacteriana de proteínas e peptídeos são realizadas. Dentre essas substancias, podemos citar as lisozimas, proteínas que hidrolisam as ligações β 1-4 glicosídicas entre o ácido N-acetilmurâmico e N-acetilglicosamina, presentes no peptidoglicano da parede celular bacteriana. Além disso, apresentam atividade de quitinase, ou seja, quebram a ligação glicosídica da quitina presente na parede fúngica. As lisozimas apresentam alta especificidade pela parede microbiana indicando aparente ausência de efeitos tóxicos aos humanos. Assim, tornando-a candidata a agente antimicrobiano em formulações cosméticas e farmacêuticas. A lisozima de ovo de galinha tem atividade antimicrobiana, entretanto não havia estudos relacionados com os micro-organismos contaminantes normalmente encontrados em produtos farmacêuticos e cosméticos. Além disso, a lisozima recombinante de Musca domestica (MdL1) não possui ainda sua atividade antimicrobiana definida. Os objetivos do trabalho foram:1) Obtenção da lisozima recombinante de Musca domestica (MdL1); 2) Avaliação a atividade antimicrobiana da MdL1 e de lisozima de ovo de galinha, Hen Egg White Lysozyme (HEWL), frente à Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 4698), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231) e Aspergillus niger (ATCC 16404); 3) Avaliação da toxicidade da lisozima em cultura de células de fibroblastos (ATCC CCL-92). A MdL1 foi obtida por meio de expressão gênica em Pichia pastoris GS115 (Invitrogen), concentrada utilizando polietilenoglicol 6000 e dialisada contra água deionizada através da membrana com porosidade seletiva de 12kDa. A homogeneidade foi analisada por eletroforese em gel de poliacrilamida em condições desnaturantes; e a atividade catalítica foi avaliada utilizando células liofilizadas de Micrococcus luteus como substrato. A atividade antimicrobiana foi determinada por método específico para cada enzima. A toxicidade in vitro das amostras foi avaliada pela viabilidade celular de fibroblastos ATCC CCL-92. A MdL1 obtida apresentou características de homogeneidade adequadas e atividade de 108,35 U/mg. A HEWL mostrou-se ativa contra S. aureus, M. luteus e C. albicans. A MdL1 apresentou-se ativa contra M. luteus, apenas. Devido a ausência de atividade antimicrobiana a MdL1 não foi submetida a avaliação citotóxica. Em relação à HEWL não demonstrou citotoxicidade na avaliação prévia realizada.
The increase in the search for natural products by pharmaceutical, cosmetic and food markets requires researches in these products development. These are directed to obtaining substances of vegetal or animal origin, as well as biotechnological products. The research in relation to antibacterial activity of proteins and peptides is carried out. Among these substances, it is possible to mention the lysozyme, protein that catalyze the break of 1,4-beta-D glucosidic bond between N-acetylmuramic acid and N-acetilglicosamine which are present in peptidoglicane of the bacterial cell wall. Besides, there is kitinase activity, that is, they break the glicosidic bond of chitin which is present in fungal wall. The lysozymes show high specificity by microbial wall indicating apparent absence of toxicological effects to human beings. Therefore, it becomes the candidate to antimicrobial ingredient in cosmetic and pharmaceutical dosage forms. The hen egg white lysozyme has antimicrobial activity, however there were no studies related to spoiled microorganism usually found in pharmaceutical and cosmetic products. In addition, there were not studies about microbial activity of recombinant Musca domestica lysozyme 1 (MdL1). The aim of this research was: 1) To obtain MdL1; 2) Evaluation of MdL1 and hen egg white lysozyme (HEWL) antimicrobial activity against Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 4698), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231) e Aspergillus niger (ATCC 16404); 3) Evaluation of lysozyme toxicity in fibroblast cells culture (ATCC CCL-92). The MdL1 was expressed as recombinant protein in Pichia pastoris GS115 (Invitrogen), concentrated using polyethylene glycol 6000 and dialyzed against deionized water through the selective porosity of 12kDa membrane. The homogeneity was analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the catalytic activity was evaluated using lyophilized cells of Micrococcus luteus as substract. The antimicrobial activity was evaluated using specific methods for each enzyme. The sample toxicity was evaluated by cell viability using ATCC CCL-92 fibroblasts. The MdL1 obtained presented suitable homogeneity characteristics and activity of 108.35 U/mg. The HEWL has showed activity against S. aureus, M. luteus e C. albicans. MdL1 only showed activity against M. luteus. Due to the absence of antimicrobial activity of MdL1 in the evaluated concentration it was not submitted to the cytotoxicity test. Regarding HEWL, it has not showed citotoxicity in the previous test.

博士
國立臺灣大學
化學工程學研究所
107
Amyloid fibril formation serves as a key pathological feature of several different human degenerative diseases. Evidence suggests that mitigation/inhibition of amyloid fibril formation is considered a promising approach toward treating these diseases. However, as of now, there has been no effective small molecule available to cure amyloid diseases. The reasons why hen egg white lysozyme was used as the model protein in this research work are as follows: (1) hen egg white lysozyme is structurally homologous to human lysozyme, which is the protein associated with hereditary systemic amyloidosis, and (2) its fibrils were found to resemble the fibrillar species of human lysozyme. Investigation of amyloid fibril formation using hen egg white lysozyme can aid in a better understanding of the possible inhibition strategies for tackling amyloid aggregation. In our study, we used three kinds of small molecules including fast green FCF, methylene blue, and brilliant blue G. Both fast green FCF and brilliant blue G are triarylmethane dyes, fast green FCF is approved by the FDA as a food dye, while brilliant blue G has been shown to be safe with good blood–brain-barrier-permeability. Methylene blue, a compound belonging to the phenothiazinium family, has the potential to treat a variety of cancerous and non-cancerous diseases with low toxicity and minimal side effects. Furthermore, evidence demonstrates that methylene blue may be a promising molecules for the treatment of Alzheimer’s disease. Here, we examine the effects of fast green FCF, methylene blue and brilliant blue G on amyloid fibril formation derived from hen egg white lysozyme using a variety of spectroscopic techniques, such as intrinsic fluorescence, ANS fluorescence and ThT fluorescence assays, transmission electron microscopy, and circular dichroism spectroscopy. ThT fluorescence intensity results show that fast green FCF(25%) and brilliant blue G(32.8%) possess better inhibition efficacy than methylene blue (below 1:1.11,ThT intensity has no significant change). However, CD results reveal that fast green FCF and brilliant blue G affect hen egg white lysozyme differently. The addition of fast green FCF was observed to reduce the β-sheet secondary structure content associated with amyloid fibrillogenesis, which was not found in the case of brilliant blue G. Our results demonstrate that the addition of brilliant blue G is not able to suppress the amyloid fibril-forming propensity of lysozyme but only shorten the length of fibrillary species. Moreover, our SDS-PAGE results suggest that more native proteins are retained in the sample. In addition, our results further suggest that the observed inhibitory actions against amyloid fibril formation is mainly correlated with the interaction between the small molecules and protein hydrophobic sites. Given that both fast green FCF and brilliant blue G have sulfonate functional groups, the two molecules are able to electrostatically interact with the protein, thus further mitigating amyloid fibril formation. In addition, we surmise that the less negative charge and two additional methyl groups attached to the triphenylmethane structure might account for why the presence of brilliant blue G induces the formation of shorter fibrils, but can not suppress amyloid fibrillogenesis. Taken together, we conclude that small molecules’ inhibitory activity toward amyloid fibril formation is dependent upon the structure, size, and types of functional groups attached to them. We believe exploring the effects of small molecule on amyloid fibrillogenesis of lysozyme can contribute to the development of drugs for the treatment of amyloid diseases.

博士
國立交通大學
應用化學系碩博士班
104
We present the first demonstration of laser trapping-controlled crystal growth of protein, which is achieved for hen egg-white lysozyme (HEWL) through a highly concentrated cluster domain formation. A continuous-wave near-infrared laser beam is employed as a trapping light source and focused at a point 10 µm away from a target HEWL crystal formed spontaneously in solution. Laser trapping of HEWL liquid-like clusters in solution increases local concentration at the focus, where liquid-liquid phase separation should be considered to be triggered. The local association and orientation of the clusters expands from the focus to its outside through the hydrogen bonding network, and a large highly concentrated domain of the clusters is formed. The target HEWL crystal is totally covered with the domain and grown depending on orientation and concentration of the clusters in the domain. First, laser trapping restricts the free motion and reorientation of the clusters in the domain, which strongly suppresses crystal growth. Subsequent laser trapping of the clusters in the initial domain continuously increases local cluster concentration, leading to change in conformation of HEWL cluster structure and the hydrogen bonding network. Consequently, the domain is transformed into another domain with lower rigidity and ordering of the clusters, which surprisingly enhances crystal growth. Moreover, the clusters in both domains have anisotropic features reflecting laser polarization, which also contributes to crystal growth. These results will enable us not only to develop a new crystal growth method but also to understand novel interactions between focused laser light and protein clusters.

碩士
朝陽科技大學
應用化學系碩士班
94
In terms of the first-order-like state transition model, we study the folding process of the egg white lysozyme by the nuclear magnetic resonance technique, and try to find out its folding core. By adjusting the concentrations of urea, DTT and other chemical reagents, the egg white lysozyme, beginning from the denaturated state, goes through an overcritical reaction path to the native conformation step by step. The characteristic of different stages in the folding process is monitored by measuring the chemistry shift indexes, the NOE signals of the backbone amino protons and α-protons, and the hydrogen-deuterium exchange rate. The result reveals that the structure of the unfolded state of lysozyme is very weak. Bringing down the concentration of DTT will induce the formation of helix conformation, further lowering the concentration of urea will stabilize the helix conformation. While urea is completely removed from the solution, β -sheet structure appears in the segment of lysozyme with sequence number from 39 to 60. However, the effect of the pH value and concentration of mannitol on the protein conformation is not obvious. According to the NMR spectroscopy, the segments C6~G22 and N2 ~F34 of the egg white lysozyme present stable helix conformation in each middle status from the unfolded state to the native conformation. Together with the result of the dynamic light scattering experiment, it is inferred that these two segments, C6~G22 and N27~F34, should be the initiating location of the folding of the egg white lysozyme.

Lysozyme is a natural antimicrobial agent that is effective against many food spoilage and pathogenic microorganisms by disintegrating their cell walls. Immobilization of lysozyme has attractive applications for use in the food industry: (1) The enzyme could be readily separated from treated foods and beverages and re-used while the foods could still be claimed additive-free; (2) It could impart stable antimicrobial capability to the surface of food packaging polymers. In this study, a novel method is described for the preparation of a highly active immobilized lysozyme system. The method addressed three key issues in the covalent attachment of a biological active protein to an insoluble support: 1.) The protein should be attached to the matrix by the fewest possible bonds to minimize conformational change; 2.) The binding site(s) on the enzyme to the supports should be located as far as practical from its active center and be nonessential for its tertiary structure; 3.) The binding method should minimize the steric interference between the support and the immobilized enzyme. Using polystyrene resin beads as support matrix, peptide spacers of various lengths composed of 6-aminocaproic acid were synthesized with the solid phase peptide synthesis method. Then the amino terminals of the spacers were derivatized with bromoacetyl bromide and coupled to the protein's only histidine residue (His-15) that is nonessential for its lytic activity. Immobilized lysozyme with a spacer composed of three 6-aminocaproic acid units displayed the best lytic result against lyophilized M. lysodeikticus cells: 2736 U/g resin with a protein load of 2.21 mg/ resin. Retained activity was 14.2% of that of the free enzyme. Preparations with longer spacers yielded higher protein load yet the retained activity remained at about 14% level. A control consisted of random coupling of lysozyme to polystyrene beads without spacer gave an activity of 158 U/G with a protein load of 1.24 mg/g resin and 1.4% of retained activity, Properties of the immobilized lysozyme system were studied, including stability, effect of pH, surface characteristics of the support. A kinetics study of the system using Eadie-Hofstee plot demonstrated strong external diffusion effects, which resulted in deviation from classic Michaelis-Menton kinetic behavior.
Graduation date: 2004

碩士
國立臺灣大學
化學工程學研究所
96
As of now, more than twenty different human proteins can fold abnormally resulting in the formation of amyloid fibril deposits and several lethal degenerative diseases, which are called amyloidoses or amyloid diseases. During the progression of the diseases, these proteins can self-assemble into stable fibrils with extensive beta-sheet conformation. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The current study is aimed at exploring the effect of varying ratio of cysteine and cystine on the fibrillogenesis of hen egg-white lysozyme. Via numerous spectroscopic techniques and transmission electron microscopy, our data revealed that the inhibition of lysozyme amyloid formation by cysteine in the redox buffer followed a concentration-dependent fashion. For instance, approximately 30 % or 70% inhibition of fibrillogenesis was observed in the presence of 1 mM or 2 mM cysteine, respectively. In contrast, the oxidized form of cysteine, cystine, nevertheless, did not influence the final level of fibrillogenesis although it lengthened the lag period and decreased the growth rate of fibril formation. Moreover, we observed in our study that the presence of cysteine led to a higher degree of disulfide bond disruption. About 53 % and 79% of disulfide bond disruptions were detected when 1 mM and 2 mM cysteine were added, respectively. It could be concluded from our results that there existed a connection between the inhibition level of fibrillogenesis and the percentage of native disulfide bond disruption for lysozyme. Our results demonstrated that the disulfide bonds could be disrupted by the addition of cysteine and thus leading to significant inhibitory effects against fibril formation. According to the results from this study and the literature, we suggested that the disulfide bridge Cys64-Cys80 which is strongly associated with lysozyme fibrillogenesis would be the last broken disulfide bond. Therefore, the fibril formation would be suppressed when this disulfide bond was broken. The outcome from this work may aid in comprehending the molecular mechanism(s) of fibrillogenesis for disulfide bond-containing amyloid proteins and development of effective therapeutics for amyloidogenic diseases.

碩士
國立交通大學
應用化學系碩博士班
103
We present the first demonstration of laser trapping-induced nucleation of hen egg white lysozyme (HEWL). When a continuous-wave near-infrared laser beam is focused into HEWL buffer solution, a millimeter-sized highly concentrated domain of the liquid-like clusters is formed around the focus. The clusters in the formed domain are strongly controlled by laser trapping at the focus and exhibit high ordering and rigidity which inhibit crystal nucleation in the domain. Actually, the nucleation has been never induced during the laser irradiation. After turning off the trapping laser, the ordering and rigidity of the cluster are relaxed. As a result, the crystals are generated densely in a few millimeter area around the focus, which is consistent with the domain size. The morphology of HEWL crystals is also much different from that on spontaneous nucleation, and the crystal quality evaluated by single X-ray crystallography is as well or better. Since the mechanism involving such a domain formation should be general for proteins, this nucleation method using laser trapping can be in principle applied to all proteins. We believe that this novel method will open a new door for controlling protein nucleation precisely.

碩士
國立臺灣大學
食品科技研究所
86
Hen egg white is the main source for commercial production of lysozyme, which can spilt the cell wall of gram-positive bacteria. Therefore, lysozyme has been broadly used in food and pharmaceutical industry as preservative or antimicrobial agent. In this study, hen egg white lysozyme was purified by using κ- carrageenan and the alcohol-insoluble solids（AIS） prepared from farm product wastes in combination with ethanol precipitation treatment, in order to establish the optimum conditions for its purificat . The purpose of this study was to provide a more economical, simple and convenient procedure for lysozyme purification on the one hand, and to promote the reuse of farm product wastes on the other. This study had established the optimum conditions of ethanol precipitation treatment of egg white lysozyme as follows: egg white was 3-fold diluted with 0.05~0.1M NaCl solution and then subjected to 30% ethanol precipitation at pH 4.0 for 5~8 hr to remove other egg white proteins. A 9 to 15-fold purification and 70~90% recovery of lysozyme were achieved. This result demonstrated that ethanol precipitation treatment is effective for the primary purification of hen egg white lysozyme. A column packed with cross-linked AIS（CL-AIS） prepared from pea pod shell could be used to chromatographically purify lysozyme directly from diluted egg white solution with an achievement of 38-fold purification and 55% recovery. This achievement is similar to the result of lysozyme purification by chromatography with a commercial ion exchanger SEPRASORB, which was chosen for comparison in this study. Besides, the CL- AIS column could also be used for further purification of the partially purified lysozym olution after ethanol precipitation treatment, and a product with 69-fold purification and 72% recovery was obtained. The addition of 0.2% κ-carrageenan to an egg white solution 3-fold diluted with distilled water to absorb lysozyme, coupled with the use of 0.5M NaCl to elute the absorbed enzyme, resulted in 6 to 17-fold purification and 70~90% recovery of lysozyme. But the use of κ-carrageenan to the partially purified enzyme solution obtained by ethanol precipitation treatment did not result in further purification of the enzyme. In batch type operation, the addition of 5% AIS to an egg white solution 3-fold diluted w distilled water, or to the supernatant after ethanol precipitation treatment to absorb lysozyme, followed by the use of 0.5M NaCl to elute the absorbed enzyme did not result in acceptable purification and recovery. The use of 0.5M NaCl／0.1 M acetic acid solution in place of 0.5M NaCl as the eluent of lysozyme for AIS, result in 4-fold purification and 74% recovery of lysozyme, which are not yet satisfactory. There was no further purification when applied to the partially purified enzyme solution after ethano l precipitation treatment.

碩士
國立交通大學
應用化學系碩博士班
104
We demonstrated a fluorescence microscopic study on formation of a large highly concentrated domain of hen egg white lysozyme (HEWL) liquid-like clusters by laser trapping. We prepared a HEWL buffer solution with a small amount of fluorescent dye-labeled HEWL molecules. A continuous wave near-infrared laser beam was used as a trapping light source and focused at a position 3 μm above a glass surface of the HEWL buffer solution for 30 min. When temporal change in fluorescence intensity in an area of 110 × 110 μm2 around the laser focus was monitored in real time under various experimental conditions, the intensity monotonously increased with the laser irradiation not only at the laser focus but also in the whole observation area. This result clearly means that a highly concentrated domain of the clusters is formed around the focus and the size is considerably larger than the focal size of about 1 μm. The further investigation of temporal change of the fluorescence intensity at a position 10 μm away from the focus showed us that laser trapping of 30 min laser irradiation produced two types of the cluster domains with different fluorescence intensity. We define these cluster domains with lower and higher fluorescence intensity as “Domain 1 and Domain 2”, respectively. In these domains, HEWL liquid-like clusters are connected with each other through hydrogen bonding interactions and form ordering and consequent rigid structure. The hydrogen bonding structure extends very rapidly up to a long distance. We systematically investigated laser polarization dependence of temporal change in the fluorescence intensity of these two domains. Interestingly, the depolarized laser irradiation produced the cluster domain with the highest fluorescence intensity. In order to gain an insight into ordering and rigidity of the clusters in two domains, we also investigated temporal change in the fluorescence intensity after stopping the laser trapping. The fluorescence intensity of Domain 1 after stopping laser trapping quickly decreased to almost an initial value for 20 min, while that of Domain 2 decreased much slowly and seemed to saturate at a certain high value for 2 hs. Surprisingly, compared to polarized laser irradiation, the fluorescence intensity in Domain 1 upon depolarized laser irradiation decreased considerably faster, in spite of the higher fluorescence intensity at 30 min of the irradiation. These results suggest that depolarized laser irradiation produces a more highly concentrated domain of the clusters with less ordering and rigidity. We also investigated growth behavior of a target HEWL crystal located in the domains with and after stopping laser trapping, and discuss dynamics and mechanism of formation and dissolution of the large highly concentrated domains of the clusters by comparing temporal change of fluorescence intensity and the crystal growth behavior.

碩士
國立臺北科技大學
化學工程系碩士班
92
In this study, molecular dynamics (MD) simulations were conducted to investigate 1) the effect of supercritical carbon dioxide on the structural integrity of hen egg white lysozyme (HEWL) and 2) the thermal unfolding behaviors of N-carbamyl-D-amino acid amidohydrolase. For the case of HEWL, 400 ps MD simulations were conduced to investigate the effect of supercritical carbon dioxide on the structural integrity of HEWL. The results indicated that HEWL reveal the ability to increase the stability of this protein in the supercritical carbon dioxide solvent. Because of the solvent was less polarity to cause hydrophobic interactions in the native structure were weakened, and local hydrogen bonds were strengthened, resulting in stabilization of the secondary structures. For N-carbamyl-D-amino acid amidohydrolase (Dcase), 1 ns molecular dynamics simulations were performed with temperature jump technique. The results show that the unfolding pathways of the two subunits of the dimeric DCase were different from each other. Subunit A lost its secondary structural integrity from the outermost layer 1 to the innermost layer 4, whereas subunit B initialed its unfolding from the interior layer 3. It indicates that the unfolding of these two subunits of Dcase may follow different pathways.

The natural ability of filamentous fungi to synthesize, glycosylate, and secrete high levels of protein products has made them potentially attractive hosts for heterologous protein production. Advances in fungal genetics enabled the expression of several high value proteins in filamentous fungi. Particularly the genus, Aspergillus has proven to be potentially useful for the expression of eukaryotic gene products. This thesis pertains to the optimization of recombinant protein production by the fungal host, Aspergillus niger. The target recombinant protein of interest is hen egg white lysozyme (HEWL). This protein encoded in the genome resulting in relatively stable gene construct; however, it is subject to extracellular protease attack. The objective of the proposed research is the development and application of engineering methodology for the analysis and optimization of a fungal bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. To accomplish this, during the first phase of this study a statistically based experimental method was used to systematically elucidate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl₂.2H₂O) on hen egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2⁵⁻¹ fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other medium components were not important within the levels tested. Then, the method of steepest ascent was employed to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g/L, peptone 34 g/L, ammonium sulfate 11.9 g/L, yeast extract 0.5 g/L, and CaCl₂.2H₂O 0.5 g/L. This medium was projected to produce theoretically 212 mg/L lysozyme. Using this optimized medium, an experimentally observed maximum lysozyme concentration of 209±18 mg/L verified the applied methodology. A second optimization approach was based on metabolic flux analysis (MFA). A comprehensive metabolic network comprising three intracellular compartments (cytoplasm, mitochondrion and peroxisome) was developed for Aspergillus niger. The metabolic flux network included carbohydrate and amino acid metabolism in both anabolic and catabolic reactions. According to experimental observations, the time course of fermentation was divided into five phases, each with unique physiological properties. The network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consists of 137 metabolites and 287 processes, of which 181 represent biochemical conversions and 106 represent transport processes between the different compartments and the extracellular environment. In addition, due to the physiological evidence some biochemical reactions considered to be active only in one direction. Linear programming was used for optimizing of the specific growth rate as the objective function in combination with 37 measured input and output fluxes of the key metabolites to evaluate corresponding intracellular flux distributions throughout the batch fermentations. The general applicability of the methodology was evaluated by establishing commonality to optimize recombinant HEWL production. The proposed model was able to predict correctly the specific growth rate, oxygen uptake rate, and carbon dioxide evolution rate with good precision. The results of the metabolic flux and sensitivity analysis were employed for medium design. Growth was biphasic; glucose was utilized initially as the carbon source and was followed by its oxidation product, gluconate, later. Logarithmic sensitivity analysis revealed that the addition of proline, alanine and glutamate benefited growth in defined media. The experimental observations and flux analysis showed that tyrosine was a potential candidate for biomass production improvement. The two amino acids, namely proline and tyrosine benefited biomass production during the initial growth phases. Glutamate and alanine were particularly important during the latter stages of the batch process. A series of growth studies were conducted with the identified amino acids added in the medium. In these preliminary nutritional experiments the contribution to growth enhancement was 46% for proline, 23% for glutamate, and 22% for tyrosine. Model predictions were further verified by conducting batch and fed-batch fermentations in a 7- liter bioreactor. The programmed addition of four amino acids (proline, glutamate, alanine, and tyrosine) according to a predetermined schedule resulted in a 44% improvement in biomass and 41% improvement in recombinant protein production. The experiments also confirmed the model prediction that extra amount of amino acids besides the identified ones would not significantly enhance biomass and the recombinant protein production. A computer-based control system was developed for the on-line monitoring and control of the major state variables (e.g., temperature, pH, and DO) during the time course of fermentation. The graphical programming environment, LabVIEW was used to acquire and integrate these variables in a supervisor computer. The temperature of the bioreactor during sterilization and fermentation was controlled using a cascade methodology. The controller parameters of the master and slave loops were determined experimentally to yield a smooth response with minimum overshoot of both the bioreactor and jacket temperatures. The program scheduled various required steps in an established order during the fermentation. This feature of the software guarantees that every necessary operation will be met. The graphical representation of the process is displayed on the screen and helps the user to follow the process and perform the required adjustments. Furthermore, different variables can be observed simultaneously and saved in text or spreadsheet files for further analysis.