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Violacein is an intensely purple pigment synthesized by various genera of bacteria that has been discovered to have a wide range of interesting biological activities which range from anticarcinogenic to antibacterial. One of the hindrances for its real-life application is that the first microorganisms found to produce the compound may act as opportunistic pathogens. Here, we report the isolation and characterization of violacein from a non-pathogenic Antarctic Iodobacter strain. Its anti-microbial properties were also tested. The method proposed here for the purification of violacein shows high yields, indicating that this Antarctic microorganism could be a valuable source for this important pigment. This is the first characterization of violacein from an Antarctic Iodobacter strain and here we also present a viable method to obtain this pigment for potential biotechnological applications.

Made available in DSpace on 2021-06-25T10:47:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 The extracellular serine protease produced by Acremonium sp. L1-4B isolated from the Antarctic continent, was purified and used for the proteolysis of bovine and caprine sodium caseinate. Protein hydrolysates were evaluated in vitro to determine their antioxidant and antihypertensive potential, and later characterized by mass spectrometry. Bovine and caprine hydrolysates produced over 24 h showed a higher content of copper chelation (25.8 and 31.2% respectively), also at this time the ABTS+• scavenging was 65.2% (bovine sample) and 67.5% (caprine sample), and bovine caseinate hydrolysate (8 h) exhibited higher iron chelation capacity (43.1%). Statistically (p < 0.05), caprine caseinate hydrolysates showed relatively higher antioxidant potential in this study. All hydrolysates showed antihypertensive potential; however peptides released from caprine caseinate after 8 h of hydrolysis were able to inhibit 75% of angiotensin-converting enzyme (ACE) activity. Nano-ESI-Q-TOF-MS/MS analysis prospected a total of 23 different peptide sequences in the bovine hydrolysate fraction, originated from the αS1- and β-casein chain, whilst in caprine hydrolysate, 31 sequences were detected, all from β-casein. The low molecular weight bovine and caprine hydrolysates obtained in this research have the potential to act in the prevention of disorders caused by oxidative reactions and in the regulation of blood pressure. These findings support the development of new functional food and nutraceutical formulations. Department of Animal Morphology and Physiology Federal Rural University of Pernambuco Department of Biochemical and Pharmaceutical Technology School of Pharmaceutical Sciences University of Sao Paulo Department of Agribusiness Food and Nutrition ESALQ University of Sao Paulo Department of Statistics and Applied Mathematics Federal University of Ceará Department of Chemistry and Environmental Science IBILCE São Paulo State University (UNESP) Department of General and Applied Biology Institute of Biosciences São Paulo State University (UNESP) Federal University of Agreste of Pernambuco Department of Chemistry and Environmental Science IBILCE São Paulo State University (UNESP) Department of General and Applied Biology Institute of Biosciences São Paulo State University (UNESP)

One new alkaloid, named as acremolin C (1), was isolated from static culture of Antarctic fungus, Aspergillus sydowii SP-1, in an investigation of the antimicrobial constituents of this Antarctic microorganism, and its structure was determined by spectroscopic methods. Additionally, four known compounds, cyclo-(L-Trp-L-Phe) (2), 4-hydroxyphenylacetic acid (3), (7S)-(+)-hydroxysydonic acid (4) and (7S, 11S)-(+)-12-hydroxysydonic acid (5), were isolated and identified. Biological studies disclosed that compounds 2, 4 and 5 showed moderate inhibitions against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) as comparing to tigecycline, while compound 1 displayed weak inhibition activities against MRSA and MRSE.

In this study, the physical factors for amylase production by Arthrobacter sp. were optimized using response surface methodology(RSM). Antarctic microorganism Arthrobacter sp. PAMC 27388 was obtained from the Polar and Alpine Microbial Collection(PAMC) at the Korea Polar Research Institute. This microorganism was confirmed for the excretion of amylase with Lugol`s solution. The amylase activity was after flask culture was as low as 1.66 mU/L before optimization. The physical factors including the inoculum volume, the initial culture pH, and the medium volume were chosen to be optimized for the enhanced amylase production. The calculated results using RSM indicate that the optimal physical factors were 2.49 mL inoculum volume, 6.85 pH and 42.87 mL medium volume with a predicted amylase production of 2.84 mU/L. The experimentally obtained amylase activity was 2.50 mU/L, which was a 150% increase compared to the level before optimization.

The Antarctic is a potentially important library of microbial resources and new bioactive substances. In this study, microorganisms were isolated from surface water samples collected from different sites of the Antarctic. 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay‐based cytotoxicity‐tracking method was used to identify Antarctic marine microorganism resources for antitumor lead compounds. The results showed that a total of 129 Antarctic microorganism strains were isolated. Twelve strains showed potent cytotoxic activities, among which a Gram‐negative, rod‐shaped bacterium, designated as N11‐8 was further studied. Phylogenetic analysis based on 16S rRNA gene sequence showed that N11‐8 belongs to the genus Bacillus. Fermented active products of N11‐8 with molecular weights of 1–30 kDa had higher inhibitory effects on different cancaer cells, such as BEL‐7402 human hepatocellular carcinoma cells, U251 human glioma cells, RKO human colon carcinoma cells, A549 human lung carcinoma cells, and MCF‐7 human breast carcinoma cells. However, they displayed lower cytotoxicity against HFL1 human normal fibroblast lung cells. However, they displayed lower cytotoxicity against HFL1 human normal fibroblast lung cells. Microscopic observations showed that the fermented active products have inhibitory activity on BEL‐7402 cells similar to that of mitomycin C. Further studies indicated that the fermented active products have high pH and high thermal stability. In conclusion, most strains isolated in this study may be developed as promising sources for the discovery of antitumor bioactive substances. The fermented active products of Antarctic marine Bacillus sp. N11‐ 8 are expected to be applied in the prevention and treatment of cancer.

Methanococcoides burtonii, a methanogen that was isolated from Ace Lake, Antarctica, has proven to be a useful model for studying the molecular mechanisms of cold adaptation in Archaea. In Ace Lake, M. burtonii only experiences temperatures below 4 degrees and nucleic acid binding proteins have been inferred to play important roles in cold adaptation. In this thesis, the three M. burtonii ctr (cold-responsive TRAM domain) genes (proteins) Mbur_0304, Mbur_0604 and Mbur_1445 are referred to as ctr1 (Ctr1), ctr2 (Ctr2) and ctr3 (Ctr3), respectively. The thesis reports the first experimental studies to assess evolutionary, biophysical and structural properties and unique characteristics related to nucleic acid binding, including specific interactions describing putative roles of Ctr3 in the cell. During purification, E. coli nucleic acids consistently co-purified with recombinant Ctr3. The liberated nucleic acid from proteins was able to be digested with RNase indicating the bound nucleic acid was RNA. The bound RNA was able to be removed by treating the recombinant proteins with mild urea to partially unfold the protein, eluting the protein across a NaCl gradient, and refolding it by dialysis. Purification of recombinant RNA-free proteins allowed thorough assessment of the structure-function-stability relationship of Ctr3. Ctr3 unfolded reversibly with a two-state mechanism (Tm of ~ 50 degrees). The predicted three-dimensional structure of Ctr3 exhibited substantial structural similarities with the TRAM domain of RumA protein (RlmD) from E. coli. The aromatic residues, particularly the four phenylalanine residues of Ctr3 appeared to be surface exposed and in close proximity to each other on the putative RNA-binding surface, similar to the aromatic residues in RumA-TRAM domain, suggesting similar roles in RNA interaction. An on-column in vitro binding assay was used to capture M. burtonii RNA targets of Ctr3, and analysed relative to a complete reconstruction of the M. burtonii transcriptome obtained from total RNA. Identification of the captured RNA using RNA-seq revealed that Ctr3 bound M. burtonii RNA with a preference for tRNA and 5S rRNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. In 5S rRNA, the motif represented one side of the stem and loop C which also appears to be solvent exposed providing possible access to Ctr3. At low temperatures, nucleic acids are prone to form stable secondary structures which consequently impede transcriptional and translational processes in the cell. In Bacteria, a family of cold shock proteins (Csp) has been postulated to resolve inhibitory structures of nucleic acids; thereby facilitating transcription and translation at low temperatures. However, while being ubiquitous in bacterial genomes, only a few csp homologs have been identified in psychrophilic Archaea; and csp genes are absent from the M. burtonii genome. The first insight into which genes in Archaea may perform an analogous function to csp genes came from proteomic analyses of M. burtonii where the increased abundance of Ctr proteins at low temperature was identified (Williams et al., 2011). The highest levels of these Ctr proteins occurred at 1 and -2 degrees and in particular, Ctr3 exhibited the highest increases (9-fold) at -2 degrees. Ctr3 and Csps both form a beta-barrel shape with anti-parallel beta-sheets that form a nucleic acid binding surface. The broad representation of single TRAM domain proteins within Archaea compared to their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins. Although, there is little sequence identity among TRAM domain and Csp proteins, based on evolutionary and tertiary structural analyses, both proteins are inferred to play important roles in cold adaptation, including low temperature translation.

Although the antioxidant capacity of carotenoids and their role in regulating membrane fluidity have been well studied, their ability to confer resistance to hypoosmotic shock is poorly understood. In this work, we analyzed the effect of a mixture of carotenoid pigments obtained from an Antarctic microorganism belonging to the genus Pedobacter on liposomal resistance to hypoosmotic conditions. Intercalation of pigments into liposomal structures resulted in an improvement of membrane resistance by decreasing the percentage of calcein released in comparison to that by liposomes without pigments. Due to these properties, such pigments could be useful for biotechnological applications.

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses.

In Antarctica microorganisms are exposed to several conditions that trigger the generation of reactive oxygen species, such as high UV radiation. Under these conditions they must have an important antioxidant defense system in order to prevent oxidative damage. One of these defenses are pigments which are part of the non-enzymatic antioxidant mechanisms. In this work we focused on the antioxidant capacity of pigments from an Antarctic microorganism belonging to Pedobacter genus. This microorganism produces different types of pigments which belong to the carotenoids group. The antioxidant capacity of a mix of pigments was analyzed by three different methods: 1,1-diphenyl-2-picrylhydrazyl, ROS detection and oxygen electrode. The results obtained from these approaches indicate that the mix of pigments has a strong antioxidant capacity. The oxidative damage induced by UVB exposure to liposomes was also analyzed. Intercalated pigments within the liposomes improved its resistance to lipid peroxidation. Based on the analysis carried out along this research we conclude that the antioxidant properties of the mix of pigments protect this bacterium against oxidative damage. These properties make this mix of pigments a powerful antioxidant mixture with potential biotechnological applications.

The glycolytic enzyme phosphoglycerate kinase (PGK) from the Antarctic microorganism Pseudomonas sp. TACII18 is a cold-adapted enzyme that displays a high specific activity at low temperatures and decreased thermostability relative to its mesophilic counterpart. Herein, the preliminary crystallization and structure solution of psychrophilic PGK in its native form and cocrystallized with 3-phosphoglyceric acid (3-PGA) and the ATP analogue adenylyl imidophosphate (AMP-PNP) is reported. The complexed form of PGK crystallized in 2-3 d at 290 K, whereas the native form of the enzyme required 8-12 months. Morphologically, both crystal forms are similar and X-ray diffraction experiments indicate that the crystals are isomorphous. The crystals diffracted to a resolution of 2.0 A and belong to the space group P3(2). with unit-cell parameters a = b = 58.5, c = 85.4 A.The glycolytic enzyme phosphoglycerate kinase (PGK) from the Antarctic microorganism Pseudomonas sp. TACII18 is a cold-adapted enzyme that displays a high specific activity at low temperatures and decreased thermostability relative to its mesophilic counterpart. Herein, the preliminary crystallization and structure solution of psychrophilic PGK in its native form and cocrystallized with 3-phosphoglyceric acid (3-PGA) and the ATP analogue adenylyl imidophosphate (AMP-PNP) is reported. The complexed form of PGK crystallized in 2-3 d at 290 K, whereas the native form of the enzyme required 8-12 months. Morphologically, both crystal forms are similar and X-ray diffraction experiments indicate that the crystals are isomorphous. The crystals diffracted to a resolution of 2.0 A and belong to the space group P3(2). with unit-cell parameters a = b = 58.5, c = 85.4 A.

The 46 kDa xylanase from the Antarctic microorganism Pseudoalteromonas haloplanktis is an enzyme that efficiently catalyzes reactions at low temperatures. Here, the crystallization of both the native protein and the SeMet-substituted enzyme and data collection from both crystals using synchrotron radiation are described. The native data showed that the crystals diffract to 1.3 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.87, b = 90.51, c = 97.23 A. SAD data collected at the peak of the selenium absorption edge proved to be sufficient to determine the heavy-atom configuration and to obtain electron density of good quality.

TAD1 is a psychrophilic strain isolated from continental frozen water in Antarctica. Study of aspartate transcarbamylase in the bacterium shows an impressive activity of this enzyme at low temperature. At 0 degree C, its activity is up to 26% of its maximal activity observed at 30 degrees C. In comparison with the Escherichia coli enzyme, some of its kinetic properties suggest that this high activity at low temperature results from an increased catalytic efficiency. This property might result from a discrete modification localized at the catalytic site, since this psychrophilic enzyme is as stable as its Escherichia coli homologue at high temperature.

Psychrophilic microorganisms, hosts of permanently cold habitats, produce enzymes which are adapted to work at low temperatures. When compared to their mesophilic counterparts, these enzymes display a higher catalytic efficiency over a temperature range of roughly 0-30 degrees C and a high thermosensitivity. The molecular characteristics of cold enzymes originating from Antarctic bacteria have been approached through protein modelling and X-ray crystallography. The deduced three-dimensional structures of cold alpha-amylase, beta-lactamase, lipase and subtilisin have been compared to their mesophilic homologs. It appears that the molecular adaptation resides in a weakening of the intramolecular interactions, and in some cases in an increase of the interaction with the solvent, leading to more flexible molecular edifices capable of performing catalysis at a lower energy cost.