José L. Copa-Patiño

The white-rot fungus Pleurotus ostreatus secretes laccase isozymes with different substrate specificities.

Four laccase isozymes (LCC1, LCC2, LCC3 and LCC4) synthesized by Pleurotus ostreatus strain V-184 were purified and characterized. LCC1 and LCC2 have molecular masses of about 60 and 65 kDa and exhibited the same pI value (3.0). Their N termini were sequenced, revealing the same amino acid sequence and homology with laccases from other microorganisms. Laccases LCC3 and LCC4 were characterized by SDS-PAGE, estimating their molecular masses around 80 and 82 kDa, respectively. By native isoelectrofocusing, their pI values were 4.7 and 4.5, respectively. When staining with ABTS and guaiacol in native polyacrilamide gels, different specificities were observed for LCC1/LCC2 and LCC3/LCC4 isozymes.

Amine and ammonium functionalization of chloromethylsilane-ended dendrimers. Antimicrobial activity studies

Novel amine- and ammonium-terminated carbosilane dendrimers of type G(n)-[Si{CH(2)O-(C(6)H(4))-3-NMe(2)}](x) or G(n)-[Si{CH(2)O-(C(6)H(4))-3-NMe(3)(+)I(-)}](x) have been synthesized and characterized up to second generation by phenolysis of (chloromethyl)silyl-terminated dendrimers with 3-dimethylamine phenol and subsequent quaternization with methyl iodide. Quaternized carbosilane dendrimers are stable in protic solvents and can be solubilised in water after the addition of less than 1% of dimethyl sulfoxide. A study of the antimicrobial activity of these cationic dendrimers of first and second generation against both gram-positive and gram-negative bacteria is also described. The results obtained demonstrate that the new ammonium-terminated carbosilane dendrimers can be considered as multivalent biocides.

Growth and release of hydroxycinnamic acids from Brewer's spent grain by Streptomyces avermitilis CECT 3339

Streptomyces avermitilis CECT 3339 is grown on Brewer’s spent grain (BSG) and the production of feruloyl esterase (FAE) and (1→4)-β-d-xylan xilanohydrolase (xylanase) activities is studied over 5 days. Maximum level of xylanases was found at day 1. FAE activity on methyl ferulate reached a maximum level at day 2, whereas FAE activity on feruloylated oligosaccharides, either from wheat bran or sugar-beet pulp, was maximal at day 1. The cultures (1–5 days) from S. avermilitis CECT 3339 grown on BSG and on other two agro-industrial residues such as de-starched wheat bran and sugar-beet pulp were tested for the release of hydroxycinnamic acids (ferulic and p-coumaric acids) from BSG. Most ferulic acid (FA) was released when culture supernatants from day 1 and BSG as carbon source (43% of total alkali-extractable ferulic acid) and from day 2 and de-starched wheat bran as carbon source (41.2%) were used. The level of p-coumaric released in all cases was lower (<9% of total alkali-extractable p-coumaric acid; pCA). The importance of the time of growth for the enzyme production involved in the hydrolysis of BSG is discussed.

Studies of the production and characterization of laccase activity in the basidiomycete coriolopsis gallica, an efficient decolorizer of alkaline effluents

Abstract The basidiomycete Coriolopsis gallica decolorizes alkaline paper effluents efficiently. In this work, we found that C. gallica produces laccase during this decolorization process. This enzymatic activity was produced in all media studied; however, the highest enzymatic activity was obtained in a medium containing paper effluent, where laccase was detected on the 2nd day of the experiment. The laccase activity of C. gallica was purified and characterized. The amino-terminal sequence of this protein showed the highest similarity with the laccase I of the basidiomycete PM1 and with Coriolus hirsutus laccase.

Carbosilane cationic dendrimers synthesized by thiol–ene click chemistry and their use as antibacterial agents

Cationic carbosilane dendrimers of generations 1–3 have been synthesized employing thiol–ene click chemistry. The obtained dendrimers present three different types of ammonium functions, two of them with the charge at the surface, –NH3+ and –NMe3+, and other with the charge internalized by the presence of ethylalcohol moieties, –[NMe2(CH2CH2OH)]+. The influence of –NMe3+ and –[NMe2(CH2CH2OH)]+ in dendrimer structure have been studied by molecular dynamics. The antibacterial properties of these families of dendrimers have been evaluated against Gram-positive (Staphylococcus aureus CECT 240) and Gram-negative (Escherichia coli CECT 515) bacterial strains, and the results have been compared with those obtained for related cationic carbosilane dendrimers functionalized by hydrosilylation reactions. These data show the relevance of the sulfur atom versus the silicon atom close to the dendrimer surface and the outer charge versus the inner charge. Finally, the stability of the most active first generation dendrimers vs. pH and temperature has also been studied.

Algoriphagus hitonicola sp. nov., isolated from an athalassohaline lagoon.

A Gram-negative, heterotrophic, aerobic, reddish-orange-pigmented, non-spore-forming, non-motile bacterial strain, designated 7-UAH(T), was isolated from salty water from the athalassohaline lagoon at El Hito, located in central Spain. The strain grew optimally at 37 degrees C and pH 7.0 and in the presence of 2.5 % NaCl. A polyphasic taxonomic study was carried out in order to characterize the strain in detail. Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain 7-UAH(T) clustered within the branch constituted by species of the genus Hongiella, which were recently transferred to the genus Algoriphagus. Analysis of the polar lipid profile and DNA G+C content also supported placement of strain 7-UAH(T) within the genus Algoriphagus. On the basis of its phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain 7-UAH(T) represents a novel species of the genus Algoriphagus, for which the name Algoriphagus hitonicola sp. nov. is proposed. The type strain is 7-UAH(T) (=CECT 7267(T) =CCM 7449(T)).

Halomonas ilicicola sp. nov., a moderately halophilic bacterium isolated from a saltern

A Gram-negative, heterotrophic, aerobic, pale orange-pigmented, non-endospore-forming and motile bacterial strain, designated strain SP8(T), was isolated from a salty water sample from the solar salterns of Santa Pola, located on the Mediterranean coast of Spain. The strain grew optimally at 37 degrees C, pH 6.5 and in the presence of 10 % NaCl. A polyphasic taxonomic study was conducted in order to characterize the strain in detail. Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain SP8(T) clustered within the branch constituted by species of the genus Halomonas. The closest phylogenetic neighbours of strain SP8(T) were Halomonas muralis LMG 20969(T) (96.0 % sequence similarity), Halomonas pantelleriensis AAP(T) (95.9 %) and Halomonas campaniensis 5AG(T) (95.8 %). Phenotypic features, the fatty acid profile and the DNA G+C content of the novel strain further supported its placement in the genus Halomonas. On the basis of phenotypic, chemotaxonomic and phylogenetic distinctiveness, it is suggested that strain SP8(T) represents a novel species for which the name Halomonas ilicicola sp. nov. is proposed. The type strain is SP8(T) (=CECT 7331(T)=CCM 7522(T)=DSM 19980(T)).

Genomes of “Spiribacter”, a streamlined, successful halophilic bacterium

BackgroundThalassosaline waters produced by the concentration of seawater are widespread and common extreme aquatic habitats. Their salinity varies from that of sea water (ca. 3.5%) to saturation for NaCl (ca. 37%). Obviously the microbiota varies dramatically throughout this range. Recent metagenomic analysis of intermediate salinity waters (19%) indicated the presence of an abundant and yet undescribed gamma-proteobacterium. Two strains belonging to this group have been isolated from saltern ponds of intermediate salinity in two Spanish salterns and were named “Spiribacter”.ResultsThe genomes of two isolates of “Spiribacter” have been fully sequenced and assembled. The analysis of metagenomic datasets indicates that microbes of this genus are widespread worldwide in medium salinity habitats representing the first ecologically defined moderate halophile. The genomes indicate that the two isolates belong to different species within the same genus. Both genomes are streamlined with high coding densities, have few regulatory mechanisms and no motility or chemotactic behavior. Metabolically they are heterotrophs with a subgroup II xanthorhodopsin as an additional energy source when light is available.ConclusionsThis is the first bacterium that has been proven by culture independent approaches to be prevalent in hypersaline habitats of intermediate salinity (half a way between the sea and NaCl saturation). Predictions from the proteome and analysis of transporter genes, together with a complete ectoine biosynthesis gene cluster are consistent with these microbes having the salt-out-organic-compatible solutes type of osmoregulation. All these features are also consistent with a well-adapted fully planktonic microbe while other halophiles with more complex genomes such as Salinibacter ruber might have particle associated microniches.

Application of the affinity binding of xylanases to oat-spelt xylan in the purification of endoxylanase CM-2 from Streptomyces chattanoogensis CECT 3336

bstract The use of the insoluble polysaccharides Avicel and oat-spelt xylan for the binding and subsequent purification of active xylanases from Streptomyces chattanoogensis was investigated. Maximum recovery of xylanases was achieved with oat-spelt xylan, using NaCl (2 M) to remove active protein. The application of this technique to the purification of xylanases resulted in the purification of an endoxylanase (CM-2) with high specific activity (729.5 U mg−1). The properties of the purified enzyme, exhibiting activity and stability between 40 °C and 60 °C and between pH 5 and 8, suggest a potential role for both the enzyme and the rapid purification protocol in the removal of hemicelluloses from kraft pulp prior to bleaching.

Production and characterization of ferulic acid esterase activity in crude extracts by Streptomyces avermitilis CECT 3339

Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates. Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast (a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage and for biopulping.