Preparation of Poly(vinyl Alcohol) Microparticles for Freeze Protection of Sensitive Fruit Crops

[Abstract] Poly(vinyl alcohol) (PVA) displays ice recrystallization inhibition (IRI) properties as many antifreeze proteins found in cold tolerant organisms. The molecular architecture and composition (molecular weight and distribution of pendant OH and acetate groups) have been studied to improve the antifreezing properties of PVA, suggesting that the molecular architecture of PVA plays an important role in IRI activity. The present work deals with the preparation of PVA microparticles using an alkaline treatment. The effect of PVA molecular weight on the morphology and antifreezeing properties of PVA microparticles was investigated. The antifreezeing property of PVA microparticles on the susceptibility of flower bud tissues to freeze damage was also evaluated. The alkaline treatment of an aqueous PVA solution produced stable polymer chain aggregates with spherical shapes. The average size of the PVA microparticles increased significantly with the increasing molecular weight of the PVA macromolecule precursor. The PVA microparticles inhibited the growth of ice crystals and blocked ice growth at concentrations as low as 0.01 % w/v. The effect of impeding ice crystal growth by preventing the joining of adjacent ice crystals is attributed to the larger size of the PVA particles adsorbed on the ice surface compared to the aggregated PVA macromolecules in saline solution. The thermal hysteresis activity of PVA macromolecules and microparticles was not detected by differential scanning calorimetry analysis. The PVA microparticles reduced the incidence of freeze injuries in flower bud tissues by 55% and their application, considering the low toxicity of PVA, has a high potential for freeze protection in fruit crops.Chile. Comisión Nacional de Investigación Científica y Tecnológica; ID16I10425Chile. Comisión Nacional de Investigación Científica y Tecnológica; ID16I20425Chile. Gobierno Regional del Biobío; R17A10003Chile. Agencia Nacional de Investigación y Desarrollo; ACE210016Chile. Agencia Nacional de Investigación y Desarrollo; ACE210012Chile. Comisión Nacional de Investigación Científica y Tecnológica; AFB170007This research was funded by projects CONICYT + FONDEF/tercer concurso IDeA en Dos Etapas del Fondo al Desarrollo Científico Y Tecnológico, FONDEF/CONICYT 2016 [grant numbers ID16I10425 and ID16I20425], CIPA, ANID Regional, GORE BIO BIO [grant number: R17A10003], ANID [grant numbers: ACE210016 and ACE210012] and CONICYT PIA/APOYO CCTE [grant number: AFB170007

Glycoside hydrolase from the GH76 family indicates that marine Salegentibacter sp. Hel_I_6 consumes alpha-mannan from fungi

Microbial glycan degradation is essential to global carbon cycling. The marine bacterium Salegentibacter sp. Hel_I_6 (Bacteroidota) isolated from seawater off Helgoland island (North Sea) contains an α-mannan inducible gene cluster with a GH76 family endo-α-1,6-mannanase (ShGH76). This cluster is related to genetic loci employed by human gut bacteria to digest fungal α-mannan. Metagenomes from the Hel_I_6 isolation site revealed increasing GH76 gene frequencies in free-living bacteria during microalgae blooms, suggesting degradation of α-1,6-mannans from fungi. Recombinant ShGH76 protein activity assays with yeast α-mannan and synthetic oligomannans showed endo-α-1,6-mannanase activity. Resolved structures of apo-ShGH76 (2.0 Å) and of mutants co-crystalized with fungal mannan-mimicking α-1,6-mannotetrose (1.90 Å) and α-1,6-mannotriose (1.47 Å) retained the canonical (α/α)6 fold, despite low identities with sequences of known GH76 structures (GH76s from gut bacteria: <27%). The apo-form active site differed from those known from gut bacteria, and co-crystallizations revealed a kinked oligomannan conformation. Co-crystallizations also revealed precise molecular-scale interactions of ShGH76 with fungal mannan-mimicking oligomannans, indicating adaptation to this particular type of substrate. Our data hence suggest presence of yet unknown fungal α-1,6-mannans in marine ecosystems, in particular during microalgal blooms

Doctor of Philosophy

dissertationOxidative folding is one of the key challenges hampering the development of peptidebased compounds as therapeutics. While disulde-rich peptides are often thought to be more appealing drug lead compounds because of their stable, highly-crosslinked structure, their oxidative folding to the correct disulde connectivity is often dicult, and is optimized in a peptide-specic way. This work advanced knowledge of chemical and biological means to improve oxidative folding of conotoxins. Herein I present a generalized folding protocol suitable for folding diverse disulde-rich peptides. I also show that the incorporation of selenocysteines to replace a disulde bridge with a diselenide eectively adds an intramolecular oxidative folding catalyst, where this bridge had previously been assumed to be static, with any folding improvements being a consequence of conformational eects. These are followed by a discussion of oxidative folding mechanisms in vivo, relating energy expenditure to directing disulde isomerization to the desired connectivity, as well as a novel analysis tool to consider the codon conservation of the cysteine residues that comprise the disulde scaold. This work represents signicant improvements to chemical strategies to eciently produce disulde-rich peptides and genetic analyses towards a better understanding of the role oxidative folding plays in the evolution of disulde scaolds of cysteine-rich peptides