Evalúa el efecto del secado convectivo en el valor nutricional, compuestos bioactivos y capacidad antioxidante in vitro del Nostoc sphaericum Vaucher ex Bornet & Flahault “cushuro” procedente de la provincia de Recuay, departamento de Áncash. Se realizó el secado del cushuro en un horno convectivo a dos temperaturas, 50 y 60°C, y velocidad de aire 3 m/s. El valor nutricional se determinó utilizando las metodologías de la AOAC, los compuestos bioactivos por espectrofotometría y la capacidad antioxidante por los métodos de captación del radical DPPH y radical catiónico ABTS•+, en muestra fresca y seca respectivamente. La muestra fresca presentó (g/100g): humedad 98,41; proteína 0,71; carbohidratos 0,55; cenizas 0,16; grasas 0,02 y fibra cruda 0,15. Asimismo, calcio 52,96; magnesio 3,34; fósforo 8,80; hierro 0,07 mg/100g; zinc 0,1 ppm y vitamina C 0,08 mg/100g. De los compuestos bioactivos: carotenos totales 4,24 mg/100g y polifenoles totales 5,48 mg EAG/100g. La capacidad antioxidante por los métodos DPPH y ABTS•+ presentaron un IC50 de 1,26 y 0,15 mg/ml respectivamente, frente al estándar Trolox. Las muestras secadas a temperaturas de 50 y 60°C presentaron (g/100g): humedad 12,74 y 9,75; proteínas 34,53 y 35,21; carbohidratos 34,59 y 35,64; cenizas 7,96 y 8,14; grasas 1,08 y 1,29; fibra cruda 9,09 y 9,96, respectivamente. Asimismo, presentaron (mg/100g): calcio 1519,36 y 1902,33; magnesio 121,15 y 116,09; fósforo 200,18 y 223,99; hierro 4,15 y 4,57, respectivamente. También, zinc 0,30 y 0,30 ppm y vitamina C 1,97 y 2,36 mg/100g, respectivamente. De los compuestos bioactivos: carotenos totales 32,21 y 123,86 mg/100g y polifenoles totales 72,63 y 122,11 mg EAG/100g, respectivamente. La capacidad antioxidante por los métodos DPPH y ABTS•+ presentaron un IC50 de 0,31 y 0,06 mg/ml e IC50 de 0,025 y 0,028 mg/ml, respectivamente, frente al estándar Trolox. Se evaluaron los resultados en base seca, obteniendo una reducción significativa (p<0,05) por efecto del secado convectivo a 50 y 60°C en el contenido nutricional a excepción de la fibra cruda y grasas que no presentaron variación y los carbohidratos que presentó un ligero incremento; así mismo en los compuestos bioactivos (carotenos totales y polifenoles totales) presentando en la muestra seca a 50°C mayor pérdida. La capacidad antioxidante registró una variación significativa (p<0,05) por efecto del secado convectivo, presentando un incremento de la capacidad antioxidante en ambas temperaturas de secado. / Tesis

This project was focused on development of extraction and purification protocol for novel cytotoxic compound isolated from soil cyanobacterium Nostoc muscorum which would allow to determine its molecular structure via NMR (nuclear magnetic resonance) and MS (mass spectrometry) techniques. Purified cytotoxin was subjected to several biochemical and microscopical experiments in order to assess its toxicological parameters, determine the mechanism of action and evaluate potential application in biotechnology or pharmacy.

Publicación a texto completo no autorizada por el autor / Compara la actividad antioxidante in vitro de los polisacáridos extracelulares de las cianobacterias Nostoc sphaericum y Nostoc commune recolectados en la laguna de Patococha, región Ancash; así como, la comparación de sus actividades. El tamizaje fitoquímico se realizó mediante los reactivos de Molish, antrona, ninhidrina, tricloruro férrico, gelatina, Shinoda, Fehling, Lieberman Bouchardat, Dragendorff, Mayer, Rosenheim, hidroxilamina, vainillin sulfúrico, Bertrand, Sonenheim y Bornträger. La actividad antioxidante de los polisacáridos se determinó por neutralización de los radicales: 1,1-difenil-2-picril-hidrazilo (DPPH) y ácido 2,2’-azinobis (3- etilbenzotiazolin)-6-sulfónico (ABTS). La comparación de las actividades antioxidantes de los polisacáridos extracelulares se realizó mediante análisis estadísticos ANOVA y T-student, utilizando el paquete IBM SPSS 24.0. En los extractos se evidenciaron presencia de carbohidratos, azúcares reductores, esteroides o triterpenos, alcaloides, catequinas y antocianinas, saponinas y antraquinonas en ambas cianobacterias. En la evaluación de la actividad antioxidante, los polisacáridos extracelulares de Nostoc sphaericum presentaron un IC50=2,068 mg/mL y un IC50=4,398 mg/mL en el ensayo de DPPH y ABTS, respectivamente, mientras que los polisacáridos extracelulares de Nostoc commune presentaron un IC50=2,482 mg/mL y un IC50=17,837 mg/mL en el ensayo de DPPH y ABTS, respectivamente. El análisis estadístico reveló que no existe diferencia significativa entre actividad antioxidante de polisacáridos extraceulares del Nostoc sphaericum y Nostoc commune medidos por el método de DPPH; sin embargo, sí existe diferencia significativa entre actividad antioxidante de los polisacáridos extracelulares de dichas especies medido por el método de ABTS. Se concluye que los polisacáridos extracelulares de Nostoc sphaericum presentaron una mayor actividad antioxidante que los polisacáridos extracelulares de Nostoc commune. / Tesis

Investigations into the nitrogen fixing apparatus in cyanobacterium Nostoc commune revealed a gene encoding for a hemoprotein, known as cyanoglobin. The cyanoglobin gene was isolated and subcloned into Escherichia coli previously. The study presented here encompasses the optimization of growth conditions for the transformed F. coli, with subsequent induction of cyanoglobin synthesis. These conditions were applied to large-scale (24-1) fermentor culture, permitting purification of approximately 200 mg cyanoglobin. Structural analyses, including absorption spectroscopy and circular dichroism, are presented. These studies indicate that cyanoglobin is a cytoplasmic hemoglobin with properties quite unlike those of leghemoglobin a and sperm whale myoglobin, which are used as references of comparison. For example, the optical spectral properties of oxycyanoglobin are different from those of leghemoglobin α and sperm whale myoglobin. In addition, the met-form of cyanoglobin has characteristics of a low-spin hemoglobin, in contrast to the high-spin met-forms of sperm whale myoglobin and leghemoglobin α. Unusually, the met- form of cyanoglobin fails to coordinate the strong-field ligands, cyanide and azide, at pH 7 and pH 9. The Soret region circular dichroism (CD) spectrum of cyanoglobin is unlike that of sperm whale myoglobin, yet is very similar to leghemoglobin α, suggesting a similar heme environment in these two hemoproteins. Far-UV CD of cyanoglobin revealed alphahelical character comparable to that of sperm whale myoglobin and leghemoglobin α. Cyanoglobin is the first monomeric hemoglobin detected in a prokaryote, raising questions concerning a possible role of cyanoglobin in early globin gene evolution. / Master of Science

A desiccation-tolerant cyanobacterium <i>Nostoc commune</i> accumulates a novel group of water stress proteins (Wsp) in response to cycles of repeated drying and rehydration. Antibodies, specific for Wsp, were used to screen a lambdafix II library of <i>N. commune</i> UTEX 584 Bam H1 DNA fragments and an 8.5-kb fragment, containing a gene cluster that synthesized a 59-kDa cross-reactive protein. The cloned fragment comprised five ORF’s. The ORF’s 59, 24, 22, 36, and 70, each potentially encode products of molecular weights of 59, 24, 22, 36, and 70-kDa, respectively. The 59 and 24 ORF products were found to be expressed in <i>E. coli</i>. The 59-kDa product of this fragment gives the strongest cross-reaction with the Wsp antiserum. The 59-kDa protein was partially purified. The 24-kDa product was successfully purified to homogeneity and partially characterized. This study used <i>E. coli</i> strain DH10B transformed with the pTrc 99A plasmid. The pTre 99A contains the 8.5-kb gene cluster fragment of interest. The products of ORF 24 and 59 were isolated using an initial 40-60 % ammonium sulfate precipitation of a clarified <i>E. coli</i> cell lysate. The clarified cell lysate was then subjected to streptomycin sulfate precipitation. The cell lysate was then dialyzed extensively. The cell lysate was then applied to a Mono Q HR 5/5 anion exchange column using a 2 M KCl gradient elution procedure. The Mono Q column yielded a fraction containing both ORF products which eluted with approximately 400 mM KCl. This fraction was then applied to a Superose 12 HR 10/30 gel filtration column. The eluent fraction containing the ORF 24 product was then reapplied to the Superose 12 to yield the final fraction containing only the ORF 24 product. The final fraction of ORF 24 was purified to homogeneity as determined by SDS-PAGE analysis. Approximately 750 μg of ORF 24 was isolated. This preparation was used for characterization studies. Characterization studies of ORF 24 consisted of an amino-terminal sequence analysis, an estimation of the molecular weight using gel filtration chromatography and SDS-PAGE analysis, and an analysis of enzymatic activity as suggested by amino acid sequence homologies. The amino-terminal sequence of ORF 24 is P V E Q R S H D. The molecular weight of ORF 24 using gel-filtration chromatography and SDS-PAGE analysis is 26-kDa and 23-kDa, respectively. From gene sequence analysis, the molecular weight of ORF 24 is known to be 24,340-Da. These data indicate that ORF 24 is a monomer. ORF 24 was found to have amino acid sequence homologies with a pectate lyase (E 4.2.2.2) periplasmic precursor from <i>Erwinia caratovora</i> subspecies and a dextransucrase (EC 2.4.1.5) precursor from <i>Streptoccocus mutans</i> GS-5. However, pectate lyase activity was not detected in cellular extracts over a 24 hour period. In addition, ORF 24 was not found to interact with 10 % substrate solutions of N-acetylglucosamine, pectin, UTEX 584 sheath material, DRH1 sheath material, sucrose, or glucose using thin layer chromatography. These studies indicate that the enzymatic activities proposed from amino acid sequence homologies have not been detected. The suggestion that ORF 24 is a water stress protein with a protective function on a structural level with regards to desiccation-tolerance requires further study. / Master of Science

Accelerated global warming plus an increasing need for energy is an equation not easily solved, thus new forms of sustainable energy production are urgently requested. In this context hydrogen production based on a cyanobacterial system offers an environmentally friendly alternative for energy capture and conversion. Cyanobacteria can produce hydrogen gas from sun light and water through the combination of photosystems and hydrogenases, and are suitable to cultivate in large scale. In the present thesis the maturation process of [NiFe]-hydrogenases is investigated with special focus on transcription of the accessory genes encoding proteins needed for assembly of the large and possibly also for the small hydrogenase subunit. The cyanobacteria used are two N2-fixing, filamentous, heterocystous strains; Nostoc sp. strain PCC 7120 and Nostoc punctiforme PCC 73102. For a biotechnological exploration of hydrogen production tools for regulatory purposes are important. The transcription factor CalA (cyanobacterial AbrB like) (Alr0946 in the genome) in Nostoc sp. strain PCC 7120 was found to be involved in hydrogen metabolism by regulating the transcription of the maturation protein HypC. Further the bidirectional hydrogenase activity was down-regulated in the presence of elevated levels of CalA, a result important to take into account when optimizing cyanobacteria for hydrogen production. CalA regulates at least 25 proteins in Nostoc sp. strain PCC 7120 and one of the down-regulated proteins was superoxide dismutase, FeSOD. The characterization of FeSOD shows that it has a specific and important function in the oxidative stress tolerance of Nostoc sp. stain PCC 7120. Since CalA is involved in regulation of both the hydrogen metabolism as well as stress responses these findings indicate that Alr0946 is an important transcription factor in Nostoc sp. strain PCC 7120 active on a global level in the cell. This thesis adds more knowledge concerning maturation and regulation of cyanobacterial hydrogenases which might be useful for future large scale hydrogen.

With accellerating global warming and pollution problems a change of energy regime is necessary. Solar energy offers a clean and unlimited energy source of enormous potential. Due to it’s intermittenet nature solar energy must be stored - ideally in the chemical bond of a carrier molecule. Hydrogen gas, H2, an energy carrier with water as only emission when used in a fuel cell, is considered to be the choise for the future. In this context cyanobacteria show promising potential as future H2 factories since they can produce H2 from solar energy and water. The main enzymes directly involved in cyanobacterial hydrogen metabolism are nitrogenases and hydrogenases. Cyanobacterial hydrogenases are either uptake hydrogenases or bidirectional hydrogenases and their maturation requires assistance of six maturation proteins and two hydrogenase specific proteases. In this thesis the transcriptional regulation, maturation and function of the cyanobacterial uptake hydrogenases were investigated in the filamentous, heterocyst forming strains Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120. Five genes, encoding proteins putatively involved in the maturation of the uptake hydrogenase were identified upstream the known maturation genes. Two transcription factors, CalA and CalB, were found interacting with the stretch of DNA forming the upstream regions of the uptake hydrogenase structural genes and the novel maturation genes. The expression of the uptake hydrogenase were  heterocysts specific and the specificity mapped to a short promoter region starting -57 bp upstream the transcription start point. In addition, the function of the uptake hydrogenase was inserted in a metabolic context. Among the proteases, a conserved region was discovered possibly involved in determining the hydrogenase specificity. This thesis has given valuable information about the transcriptional regulation, maturation and function of the uptake hydrogenase in filamentous, heterocystous cyanobacteria and identified new targets for bioengineering of mutant strains with higher H2 production rates.

<p>Phylogeny and morphology were examined for the cyanobionts from the water fern <i>Azolla </i>and the cyanobacterial genus<i> Nostoc</i> originating from symbioses with different host plants (genera <i>Gunnera, Cycas,</i> <i>Dioon,</i> <i>Encephalarthos, Macrozamia, </i>and <i>Anthoceros</i>), the lichen genus<i> Pannaria</i>, and free-living <i>Nostoc</i> isolates from different habitats. <i>Nostoc</i> isolates of <i>Pannaria</i> formed a closely related group, but, in general, no monophyletic nature was attributed to the genus <i>Nostoc</i>, in contrast to the cyanobionts from <i>Azolla </i>which were contained in a unique monophyletic group. No correlation was detected between the diversity of the studied cyanobacteria and their geographical origin, while high host specificity was proved for the <i>Azolla</i> cyanobionts and the <i>Nostoc</i> isolates from the bipartite <i>Pannaria</i> lichen. Two patterns of evolution leading to symbiotically competent heterocystous cyanobacteria were distinguished, one comprising symbiotic <i>Nostoc </i>species and the other comprising cyanobacteria in association with the water fern <i>Azolla</i>.</p><p>The production of the non-protein amino acid BMAA, a potential neurotoxin, was also examined. A rapid and sensitive method involving the lysis and extraction of amino acids from cyanobacteria combined with an HPLC assay for fluorescence detection of BMAA was developed. To determine whether the plant or the cyanobacterium was the origin of the BMAA in the cyanobacterium-<i>Azolla </i>symbiosis, the cyanobacterium-free <i>Azolla pinnata</i> var <i>imbricata </i>strain 511 was examined. HPLC analysis demonstrated a significant BMAA production in the absence of the cyanobacterium. However, PCR and cloning revealed the presence of bacteria of the genus <i>Ochrobactrum </i>in the plant.</p>

Phylogeny and morphology were examined for the cyanobionts from the water fern Azolla and the cyanobacterial genus Nostoc originating from symbioses with different host plants (genera Gunnera, Cycas, Dioon, Encephalarthos, Macrozamia, and Anthoceros), the lichen genus Pannaria, and free-living Nostoc isolates from different habitats. Nostoc isolates of Pannaria formed a closely related group, but, in general, no monophyletic nature was attributed to the genus Nostoc, in contrast to the cyanobionts from Azolla which were contained in a unique monophyletic group. No correlation was detected between the diversity of the studied cyanobacteria and their geographical origin, while high host specificity was proved for the Azolla cyanobionts and the Nostoc isolates from the bipartite Pannaria lichen. Two patterns of evolution leading to symbiotically competent heterocystous cyanobacteria were distinguished, one comprising symbiotic Nostoc species and the other comprising cyanobacteria in association with the water fern Azolla. The production of the non-protein amino acid BMAA, a potential neurotoxin, was also examined. A rapid and sensitive method involving the lysis and extraction of amino acids from cyanobacteria combined with an HPLC assay for fluorescence detection of BMAA was developed. To determine whether the plant or the cyanobacterium was the origin of the BMAA in the cyanobacterium-Azolla symbiosis, the cyanobacterium-free Azolla pinnata var imbricata strain 511 was examined. HPLC analysis demonstrated a significant BMAA production in the absence of the cyanobacterium. However, PCR and cloning revealed the presence of bacteria of the genus Ochrobactrum in the plant.

Today, mankind faces a new challenge in energetic terms: a new Industrial Revolution is imperative, already called by some as an Energetic Revolution. This corresponds to a conversion to clean, environmentally friendly and renewable energy sources. In this context, hydrogen arises as a valid alternative, since its combustion produces a considerable amount of energy and releases solely water as a by-product. In the present thesis, two model cyanobacteria, namely Synechocystis sp. strain PCC 6803 and Anabaena/Nostoc sp. strain PCC 7120, were used to examine the hydrogen metabolism. The efforts were focused on to understand the transcription regulation of the hox genes, encoding the structural elements of the bidirectional hydrogenase enzyme. Here, it is shown that such regulation is operated in a very distinct and intricate way, with different factors contributing to its delicate tuning. While in Synechocystis sp. strain PCC 6803 the hox genes were shown to be transcribed as a single operon, in Anabaena/Nostoc sp. strain PCC 7120 they were shown to be transcribed as two independent operons (possibly three). Two transcription factors, LexA and AbrB-like protein, were identified and further characterized in relation to the hydrogen metabolism. Furthermore, different environmental conditions were demonstrated to operate changes on the transcription of the bidirectional hydrogenase genes. In addition, functional studies of three open reading frames found within the hox operon of Synechocystis sp. strain PCC 6803 suggest that this may be a stress responsive operon. However, based on the gained knowledge, it is still not possible to connect the signal transduction pathways, from the environmental signal, through the response regulator, to the final regulation of the hox genes. Nevertheless, the crucial importance of studying the transcription regulation of the different players involved in the hydrogen metabolism is now established and a new era seems to be rising.