Extra-Cytoplasmic Function (ECF) σ factors are a subset of σ factors many organisms use to transcribe specific genes in response to environmental cues. In the absence of an inducing signal, ECF σ factors are inhibited by an anti-σ factor that prevents the ECF σ factor from interacting with RNA polymerase. The ECF σ factor σV from Bacillus subtilis is activated in response to lysozyme stress. Lysozyme damages bacterial peptidoglycan by cleaving at the 1,4-ß-linkage between N-acetylmuramic acid and N-acetylglucosamine. In the absence of lysozyme, the activity of σV is inhibited by the anti-σ factor RsiV, a single pass transmembrane protein. The two main components of this project have been to elucidate the mechanism of σV activation and examine how this system senses lysozyme stress. In chapter 2 we show that the activation of σV is specific to lysozyme, and the anti-σ factor RsiV is degraded by a step wise proteolytic cascade known as Regulated Intramembrane Proteolysis (RIP). In the presence of lysozyme, the extracellular domain of RsiV is removed by cleavage at site-1. Upon removal of the extracellular domain, the site-2 protease, RasP, cleaves RsiV within the membrane. The remainder of RsiV is degraded by cytosolic proteases allowing σV to interact with RNA polymerase. In response to lysozyme stress σV activates an O-actyltransferase, OatA, which modifies the peptidoglycan to prevent further lysozyme damage. Our studies in chapter 3 identifed the protease(s) responsible for site-1 cleavage of RsiV and revealed RsiV directly interacts with lysozyme. We determined the cleavage site of the site-1 protease using N-terminal sequencing, and demonstrate that disruption of site-1 cleavage blocks σV activation. Site-1 cleavage occurs at what appears to be a signal peptide cleavage site. We demonstrate that four out of the five signal peptidases from B. subtilis are able to cleave RsiV at site-1 in vitro only in the presence of lysozyme. Additionally, we show that the extracellular domain of RsiV directly binds the inducing substrate lysozyme. In chapter 4 we focus on determining if the interaction between RsiV and lysozyme is necessary for σV activation. Based on the co-crystal structure of RsiV and lysozyme we mutated sveral residues predicted to be involved in binding. One combination of RsiV mutations (S169W, P259A, Y261A) was unable to activate σV and subsequently was unable to bind lysozyme. We propose a RIP dependent mechanism of σV activation that is contingent upon the anti-σ factor (RsiV) directly binding the inducing signal (lysozyme) to present the site-1 cleavage site to signal peptidase. The co-crystal structure of RsiV and lysozyme also revealed that RsiV interacts with the active site of lysozyme. We demonstrate that purified RsiV inhibits lysozyme activity suggesting RsiV provides an additional lysozyme response mechanism. Thus, the anti-σ factor RsiV senses the presence of lysozyme, activates σV, and protects against further lysozyme damage.

Purpose To study the impact of lactoferrin and lipids on the kinetic deposition and denaturation of lysozyme on contact lens materials. Methods The contact lenses investigated in this thesis included two silicone hydrogel lenses [AIR OPTIX AQUA; lotrafilcon B and ACUVUE OASYS; senofilcon A] and two conventional hydrogel lenses [ACUVUE 2; etafilcon A and PROCLEAR; omafilcon A]. All lenses were incubated in four solutions: a complex artificial tear solution (ATS); an ATS without lactoferrin; an ATS without lipids; and an ATS without lactoferrin and lipids. Following various time points, all lenses were prepared for lysozyme analysis using the methods below: • To quantify the kinetic uptake of lysozyme to different contact lens materials, I125-radiolabelled lysozyme was added to each incubation solution. Total lysozyme deposition was quantified using a gamma counter. • To study the activity of lysozyme deposited to contact lenses, a fluorescence-based lysozyme activity assay was compared to a turbidity assay. Potential interactions with lens materials and extraction solvents were evaluated. • To investigate the kinetic denaturation of lysozyme deposited to different contact lens materials, the fluorescence-based activity assay and the enzyme-linked immunosorbent assay were used. Results The presence of lactoferrin and lipids decreased lysozyme uptake to lotrafilcon B. Lysozyme deposition on senofilcon A was greater in the absence of lipids after day 21, however the opposite was seen with etafilcon A, where lysozyme uptake was lower without lipids in the ATS. Lactoferrin and/or lipids had no effect on lysozyme adsorption to omafilcon A. The fluorescence-based lysozyme activity assay demonstrated high sensitivity and a wide linear range of detection, which covers the amount of lysozyme typically extracted from contact lenses. Using this assay, lysozyme activity on both silicone hydrogel materials was lower in the presence of lipids in the ATS. In addition, lactoferrin had a protective effect on lysozyme activity for lysozyme sorbed to senofilcon A. Moreover, the presence of lactoferrin and/or lipids did not exhibit any effect on lysozyme denaturation with conventional hydrogel lenses. Conclusions The presence of lactoferrin and lipids in an artificial tear solution impacted lysozyme deposition and denaturation of lysozyme on various contact lenses. It is important for in vitro studies, when developing tear film models, to consider the effects of tear film components when investigating protein deposition and denaturation on contact lenses.

This study investigated some functional and biochemical characteristics of a bacteriolytic protein in the salivary glands of adult Aedes aecupti. A method for the isolation of this protein from other mosquito salivary gland components is also described. Based on some of its biochemical properties, this bacteriolytic protein can be classified as a lysozyme. This protein is strongly-linked to mosquito sugarfeeding activity because there is a statistically significant (P < 0.05) increase in the levels of lytic activity six hours before mosquitoes start to sugar feed. By its bacteriolytic action, it may function as a protective mechanism against bacteria-contaminated sugar meals. Preliminary work suggests that mosquitoes exposed to lyophilized Micrococcus lysodeikticus in their sugar meal respond by increasing the lytic activity in their salivary glands. The levels of bacteriolytic activity are apparently not affected by bloodfeeding. In the absence of feeding, as in teneral and bloodfed mosquitoes, salivary bacteriolytic activity increases to a maximum, then levels off. This suggests a regulation of the synthesis of this salivary protein that is independent of the feeding state of the adult mosquito. A combination of centrifugation, polyacrylamide gel electrophoresis (non-denaturing and denaturing), cation exchange chromatography and gel filtration, was used to isolate the protein from other mosquito salivary gland components. This salivary protein is lysozyme-like in several aspects: 1) it lyses bacterial cell walls of M. lysodeikticus, 2) it is a basic protein with a pI between 7.47 and 8.89, 3) it is thermostable at low pH, and loses its activity at high pH, and 4) it is composed of one polypeptide chain. Its molecular weight is twice that of hen egg white lysozyme. This salivary bacteriolytic protein is the first insect exocrine lysozyme to be characterized. / Graduation date: 1992

NO

Efficient separation of target protein from impurities is crucial in bioseparation for large scale production and purity of the target protein. Two separation process approaches were considered in this study. The first approach focused on identifying major impurity and optimization of solution properties for target protein purification. The second approach consisted of designing an adsorbent that interacted specifically with the target molecule. The first study included modification of protein solution properties (pH, ionic strength, buffer ions) in order to maximize lysozyme purification by a strong cation exchange resin. The interaction of phytic acid, a major impurity, present in transgenic rice extracts, that contributes to decreased lysozyme adsorption capacity on SP Sepharose was evaluated. The target protein was lysozyme, which is used in a purified form as a baby formula additive to reduce gastrointestinal tract infections. At constant ionic strength, lysozyme in pH 4.5 acetate buffer had a higher binding capacity and stronger binding strength than at pH 6.0. Lysozyme in sodium phosphate buffer of pH 6.0 exhibited lower adsorption capacity than in pH 6 Tris buffer. Binding capacity and strength were significantly affected by phytic acid in all studies buffers. The second study consisted of surface modification of microfiltration membranes for protein purification and separation and reduces fouling. This study describes adsorption and fouling of chemically modified microfiltration membranes with bovine serum albumin (BSA) and immunoglobulin G (IgG). Least fouling resulted with polyethylene glycol (PEG) membranes when BSA protein was used. Amine-functionalized membranes showed specific interaction with BSA. There was multi-layer deposition of IgG on amine-functionalized membrane. G3 membrane synthesized to selectively bind IgG seemed a noble option to separate IgG from a protein mixture.

SAR endolysins are a recently discovered class of muralytic enzymes that are regulated by dynamic membrane topology. They are synthesized as enzymatically inactive integral membrane proteins during the phage infection cycle and then are activated by conformational remodeling upon release from the membrane. This topological duality depends on N-terminal SAR (Signal-Anchor-Release) domains, which are enriched in weakly hydrophobic residues and require the proton motive force to be maintained in the bilayer. The first SAR endolysin to be characterized was P1 Lyz, of phage P1. Its activation requires a disulfide bond isomerization involving its catalytic Cys initiated by a free cysteine thiol from the newly-liberated SAR domain. A second mode of disulfide bond regulation, as typified by Lyz103 of the Erwinia Amylovora phage ERA103, has been demonstrated. In its membrane bound form, Lyz103 is inactivated by a disulfide that is formed between cysteine residues flanking a catalytic glutamate. A second class of SAR endolysins, typified by R21, the lysozyme of the lambdoid phage 21, does not require disulfide bond isomerization for activation. Rather, these proteins are dependent on the release of the SAR domain for proper folding of the catalytic cleft. Bioinformatic analysis indicates that the regulatory theme of R21 is common in the SAR endolysins of dsDNA phages. Furthermore, bioinformatic study of endolysins of dsDNA phage of Gram-negative hosts revealed two new classes of SAR endolysins that are not homologous to T4 gpe, as all SAR endolysins were once thought to be. SAR endolysins were found in nearly 25% of sequenced dsDNA phages of Gram- negative hosts including 933W, which is involved in the release of Shiga toxin from EHEC strain EDL933. An inhibitor study against the SAR endolysin of 933W, R933W, was performed using a custom compound library in a high through-put, in vivo lysis assay. Of nearly 8,000 compounds screened, one compound, designated 67-J8, inhibited lysis but not growth. In vivo and in vitro experiments show that the compound has no effect on R933W activity, accumulation, or secretion. In vivo experiments suggest that 67-J8 increases the proton motive force, thereby presumably retaining the SAR domain in the membrane.

This thesis describes an investigation of the folding and stability of a series of derivatives of the proteins lysozyme and α-lactalbumin which lack one or more of their four native disulphide bridges. Removal of the disulphide bridge which links the N- and C-termini from hen lysozyme results in a three-disulphide derivative (CM^6,127-lysozyme). This has a profound effect on its stability against thermal denaturation, the T_m for unfolding being reduced by 25°C at pH 3.8. Calorimetric measurements performed on this three-disulphide derivative indicate that this reduction in stability may be attributed entirely to an increase in the entropy difference between the native and denatured states. Kinetic refolding studies of CM^6,127-lysozyme using stopped flow optical methods and hydrogen exchange pulse labelling in conjunction with NMR and electrospray ionisation mass spectrometry (ESI-MS) suggest that this reduced stability manifests itself primarily in the α-domain of the protein. A transient intermediate populated during refolding of the unmodified protein can no longer be detected during folding of the derivative resulting in highly cooperative folding under the conditions investigated. The structure and stability of a three- and two-disulphide derivative of the homologous protein, α-lactalbumin have been investigated by NMR spectroscopy. The three-disulphide species, like its lysozyme counterpart, can adopt native structure but this is much more unstable than the intact protein. Removal of a second disulphide bridge, however, destabilises α-lactalbumin to the extent that the native state is no longer formed. Instead, in the presence of Ca²⁺ and high concentrations of salt, a partially structured state is induced which has some elements of tertiary structure present. Novel techniques of ESI-MS have been developed to study protein folding and stability using hydrogen exchange techniques. Applications to the investigation of cooperativity in protein folding, stability in native, partially folded and unfolded states, and the interactions of a partially folded protein with the chaperone GroEL are described.

Purpose To study the impact of lactoferrin and lipids on the kinetic deposition and denaturation of lysozyme on contact lens materials. Methods The contact lenses investigated in this thesis included two silicone hydrogel lenses [AIR OPTIX AQUA; lotrafilcon B and ACUVUE OASYS; senofilcon A] and two conventional hydrogel lenses [ACUVUE 2; etafilcon A and PROCLEAR; omafilcon A]. All lenses were incubated in four solutions: a complex artificial tear solution (ATS); an ATS without lactoferrin; an ATS without lipids; and an ATS without lactoferrin and lipids. Following various time points, all lenses were prepared for lysozyme analysis using the methods below: • To quantify the kinetic uptake of lysozyme to different contact lens materials, I125-radiolabelled lysozyme was added to each incubation solution. Total lysozyme deposition was quantified using a gamma counter. • To study the activity of lysozyme deposited to contact lenses, a fluorescence-based lysozyme activity assay was compared to a turbidity assay. Potential interactions with lens materials and extraction solvents were evaluated. • To investigate the kinetic denaturation of lysozyme deposited to different contact lens materials, the fluorescence-based activity assay and the enzyme-linked immunosorbent assay were used. Results The presence of lactoferrin and lipids decreased lysozyme uptake to lotrafilcon B. Lysozyme deposition on senofilcon A was greater in the absence of lipids after day 21, however the opposite was seen with etafilcon A, where lysozyme uptake was lower without lipids in the ATS. Lactoferrin and/or lipids had no effect on lysozyme adsorption to omafilcon A. The fluorescence-based lysozyme activity assay demonstrated high sensitivity and a wide linear range of detection, which covers the amount of lysozyme typically extracted from contact lenses. Using this assay, lysozyme activity on both silicone hydrogel materials was lower in the presence of lipids in the ATS. In addition, lactoferrin had a protective effect on lysozyme activity for lysozyme sorbed to senofilcon A. Moreover, the presence of lactoferrin and/or lipids did not exhibit any effect on lysozyme denaturation with conventional hydrogel lenses. Conclusions The presence of lactoferrin and lipids in an artificial tear solution impacted lysozyme deposition and denaturation of lysozyme on various contact lenses. It is important for in vitro studies, when developing tear film models, to consider the effects of tear film components when investigating protein deposition and denaturation on contact lenses.

Thesis (Master)--İzmir Institute of Technology, İzmir, 2005. / Keywords: Biopreservatives, antimicrobial enzymes, antioxidant proteins, edible films, functional packaging materials. Includes bibliographical references (leaves.88-101).

Proefschrift Rijksuniversiteit Groningen. / Omslagtitel: The soluble lytic transglycosylase family of Escherichia coli. Datum laatste controle: 06-05-97. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.