The purpose of this study is to develop the production method of CO2 hydrate-slurry. In this paper, the production process of CO2 hydrates with pure water dissolved antifreeze proteins (AFPs) is discussed. CO2 hydrate-slurry can be transported from a production place to storage one with a small pressure loss. The AFPs have made the hydrate particles be small and well disperse. It is revealed that the Type III AFPs are effective for the inhibition of structure I hydrate production. By the present experiments, the induction time for the hydrate production increases, and moreover the formation rate of the hydrate and the increasing rate of an agitator torque decrease.

Ice-associating proteins (IAPs) are proteins that interact directly with ice crystals, either by offering a site for nucleation, i.e. ice nucleating proteins (INPs), or by binding to nascent crystals to prevent addition of more water molecules, i.e. antifreeze proteins (AFPs). AFPs have been found to inhibit the formation of clathrate-hydrates, ice-like crystalline solids composed of water-encaged guest molecules. Study of AFP-hydrate interaction is leading to a greater understanding of AFP adsorption and of the mechanism behind the “memory effect” in hydrates, wherein previously frozen crystals reform more quickly after a brief melt. AFP is currently the only known memory inhibitor. Such a low-dosage hydrate inhibitor (LDHI) is of great interest to the oil and gas industry, as hydrate formation and reformation in the field is a huge problem. Bacterial AFPs, though largely uncharacterized, may be the best candidates for large-scale production of hydrate inhibitors, given the difficulties in obtaining AFP from other sources. The popular kinetic inhibitors (KIs) polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap) were used for points of comparison in experiments exploring the hydrate-inhibition activity of several ice-associating bacteria and proteins. The addition of the soil microbe, Chryseobacterium, increased the average lag-time to tetrahydrofuran (THF) hydrate formation by 14-fold, comparable to PVP or PVCap. Samples containing Pseudomonas putida, a bacterium having both ice-nucleation protein (INP) and AFP activity, had lag-times double that of the control. Solutions with P. putida and Chryseobacterium sometimes formed hydrate slurries of stunted crystal nuclei instead of solid crystals. No inhibition of memory or nucleation was noted in bacterial assays, however bacteria with INP activity was linked to unusually rapid memory reformation. Quartz crystal microbalance experiments with dissipation (QCM-D) showed that a tight adsorption to SiO2 and resistance to rinsing are correlated with a molecule’s inhibition of hydrate formation and reformation. These results support a heterogeneous nucleation model of the memory effect, and point to the affinity of AFP for heterogeneous nucleating particles as an important component of memory inhibition. / Thesis (Master, Biology) -- Queen's University, 2008-05-30 15:20:38.749

Antifreeze proteins (AFPs) protect marine teleosts from freezing in icy seawater by binding to nascent ice crystals and preventing their growth. It has been suggested that the gene dosage for AFPs in fish reflects the degree of exposure to harsh winter climates. The starry flounder, _Platichthys stellatus_, has been chosen to examine this relationship because it inhabits a range of the Pacific coast from California to the Arctic. This flatfish is presumed to produce type I AFP, which is an alanine-rich, amphipathic alpha-helix. Genomic DNA from four starry flounder was Southern blotted and probed with a cDNA of a winter flounder liver AFP. The hybridization signal was consistent with a gene family of approximately 40 copies. Blots of DNA from other starry flounder indicate that California fish have far fewer gene copies whereas Alaska fish have far more. This analysis is complicated by the fact that there are three different type I AFP isoforms. The first is expressed in the liver and secreted into circulation, the second is a larger hyperactive dimer also thought to be expressed in the liver, and the third is expressed in peripheral tissues. To evaluate the contribution of these latter two isoforms to the overall gene signal on Southern blots, hybridization probes for the three isoforms were isolated from starry flounder DNA by genomic cloning. Two clones revealed linkage of genes for different isoforms, and this was confirmed by genomic Southern blotting, where hybridization patterns indicated that the majority of genes were present in tandem repeats. The sequence and diversity of all three isoforms was sampled in the starry flounder genome by PCR. All coding sequences derived for the skin and liver isoforms were consistent with the proposed structure-function relationships for this AFP, where the flat hydrophobic side of the helix is conserved for ice binding. There was greater sequence diversity in the skin and hyperactive isoforms than in the liver isoform, suggesting that the latter evolved recently from one of the other two. The genomic PCR primers are currently being used to sample isoform diversity in related right-eyed flounders to test this hypothesis. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-30 13:38:08.346

Antifreeze proteins (AFPs) are produced by a variety of organisms to either protect them from freezing or help them tolerate being frozen. Recent structural work has shown that AFPs bind to ice using ordered surface waters on a particular surface of the protein called the ice-binding site (IBS). These 'anchored clathrate' waters fuse to particular planes of an ice crystal and hence irreversibly bind the AFP to its ligand. An AFP isolated from the perennial ryegrass, Lolium perenne (LpAFP) was previously modelled as a right-handed beta helix with two proposed IBSs. Steric mutagenesis, where small side chains were replaced with larger ones, determined that only one of the putative IBSs was responsible for binding ice. The mutagenesis work also partly validated the fold of the computer-generated model of this AFP. In order to determine the structure of the protein, LpAFP was crystallized and solved to 1.4 Å resolution. The protein folds as an untwisted left-handed beta-helix, of opposite handedness to the model. The IBS identified by mutagenesis is remarkably flat, but less regular than the IBS of most other AFPs. Furthermore, several of the residues constituting the IBS are in multiple conformations. This irregularity may explain why LpAFP causes less thermal hysteresis than many other AFPs. Its imperfect IBS is also argued to be responsible for LpAFP's heightened ice-recrystallization inhibition activity. The structure of LpAFP is the first for a plant AFP and for a protein responsible for providing freeze tolerance rather than freeze resistance. To help understand what constitutes an IBS, a non-ice-binding homologue of type III AFP, sialic acid synthase (SAS), was engineered for ice binding. Point mutations were made to the germinal IBS of SAS to mimic key features seen in type III AFP. The crystal structures of some of the mutant proteins showed that the potential IBS became less charged and flatter as the mutations progressed, and ice affinity was gained. This proof-of-principle study highlights some of the difficulties in AFP engineering. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2012-07-18 15:24:42.082

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

The study of marine antifreeze proteins has provided new findings. The blue mussel (Mytilus edulis) was known to have antifreeze activity; however, the antifreeze protein or other molecule responsible has never been characterized. Activity was evident in mussels from each of the Maritime provinces, Canada. The antifreeze molecule was shown to alter ice crystal morphology. It functioned over a wide range of pH values and it showed protease resistance. Nonetheless, its purification was not achieved. A winter flounder (Pseudopleuronectes americanus) ?-helical antifreeze protein, wflAFP6, has been shown to form amyloid-like fibrils during freezing. Separation of different aspects of the freezing process demonstrated that equilibrium freezing with an ice template is necessary for conversion of the wflAFP6 to the amyloid-like conformation. Amyloid-like conformation was determined by dye binding and electron microscopy. The effects of wflAFP6 concentration and solution properties were determined in order to better understand the process of conversion.

One of the largest problems in current medicine is the shortage of organs for transplant due to technological limitations in the storage of organs for any length of time. A possible solution to this problem would involve cryopreservation. However, current cryopreservatives such as sucrose or DMSO have concerning cytotoxic issues that limit their possible applications. A major cause of cryoinjury is the uncontrolled recrystallization of inter and intra-cellular ice crystals that occurs during the thawing process leading to mechanical damage and dehydration. The Ben lab has thus been interested in the design of compounds that are capable of inhibiting this process but do not possess other undesirable properties found in the native compounds. These synthetic analogues have been shown to increase cellular viability post-thaw. A series of mixed α/β glycopeptides are prepared and analyzed for antifreeze properties. The results of this study imply that it is not the gross conformation of the glycopeptide that is responsible for activity, but rather that intramolecular relationships may be responsible for disrupting the reorganization of ice. A technique was devised for the incorporation of triazoles into the analogues to investigate the importance of the linker and to greatly simplify the synthesis of a library of glycoconjugates. It was found that the IRI activity of glycopeptides is very sensitive to the distance between carbohydrate and peptide backbone. The electron density at the anomeric oxygen is an important parameter with respect to intramolecular networks. A series of substituted galactosides is presented that modify the electronics of the anomeric oxygen. The results demonstrate that decreasing electron density at this position appears to improve IRI activity in a predictable manner. To better understand the remarkable IRI activity of a key analogue, it was systematically truncated. This study led to the serendipitous discovery of a series of very highly IRI active analogues that do not contain a peptide backbone. These compounds represent the first non-glycopeptides that can show very significant IRI activity even at very low concentrations. The final portion of the thesis reports the efforts towards the preparation of a carbasugar analogue of AFGP-8.

Mémoire (M.Eng.)--Université du Québec à Chicoutimi, 1995. / Document électronique également accessible en format PDF. CaQCU

Mémoire (M.Eng.)--Université du Québec à Chicoutimi, 1997. / Document électronique également accessible en format PDF. CaQCU

Mémoire (M.Eng.)--Université du Québec à Chicoutimi, 1999. / Document électronique également accessible en format PDF. CaQCU