Paola Di Donato

Synthesis, Production, and Biotechnological Applications of Exopolysaccharides and Polyhydroxyalkanoates by Archaea

Extreme environments, generally characterized by atypical temperatures, pH, pressure, salinity, toxicity, and radiation levels, are inhabited by various microorganisms specifically adapted to these particular conditions, called extremophiles. Among these, the microorganisms belonging to the Archaea domain are of significant biotechnological importance as their biopolymers possess unique properties that offer insights into their biology and evolution. Particular attention has been devoted to two main types of biopolymers produced by such peculiar microorganisms, that is, the extracellular polysaccharides (EPSs), considered as a protection against desiccation and predation, and the endocellular polyhydroxyalkanoates (PHAs) that provide an internal reserve of carbon and energy. Here, we report the composition, biosynthesis, and production of EPSs and PHAs by different archaeal species.

Metal ions as potential regulatory factors in the biosynthesis of red hair pigments: a new benzothiazole intermediate in the iron or copper assisted oxidation of 5-S-cysteinyldopa

In the presence of iron or copper ions, the course of the oxidation in air of 5-S-cysteinyldopa (1), the main biosynthetic precursor of pheomelanins and trichochromes, was markedly changed affording two main products. One of these was identified as the oxobenzothiazine 8, previously obtained under nonphysiologically relevant conditions, while the other was characterized as the novel hydroxybenzothiazole 9. Besides 8 and 9, carboxylated and noncarboxylated benzothiazine products were obtained by persulfate oxidation of 1 in the presence of iron or copper ions. The ratio of formation yields of carboxylated/noncarboxylated benzothiazines, determined after reduction of the mixture, was lower than that of the control reaction run in the absence of metal ions, and much lower than that of the oxidation carried out in the presence of zinc ions, in agreement with a recent report. Notably, 8 and 9 were formed in variable yields under different oxidation conditions including tyrosinase/O(2), peroxidase/hydrogen peroxide, and the hydrogen peroxide/or (9E,11Z,13S)-13-hydroperoxyoctadeca-9,11-dienoic acid/Fe(III) systems. Mechanistic routes to 8 and 9 were proposed based on the results of experiments involving in situ generation of labile benzothiazine intermediates. Overall, these results allow to formulate an improved biosynthetic scheme in which metal ions act as critical regulatory factors determining pheomelanin vs. trichochromes formation.

Fermentation technologies for the optimization of marine microbial exopolysaccharide production.

In the last decades, research has focused on the capabilities of microbes to secrete exopolysaccharides (EPS), because these polymers differ from the commercial ones derived essentially from plants or algae in their numerous valuable qualities. These biopolymers have emerged as new polymeric materials with novel and unique physical characteristics that have found extensive applications. In marine microorganisms the produced EPS provide an instrument to survive in adverse conditions: They are found to envelope the cells by allowing the entrapment of nutrients or the adhesion to solid substrates. Even if the processes of synthesis and release of exopolysaccharides request high-energy investments for the bacterium, these biopolymers permit resistance under extreme environmental conditions. Marine bacteria like Bacillus, Halomonas, Planococcus, Enterobacter, Alteromonas, Pseudoalteromonas, Vibrio, Rhodococcus, Zoogloea but also Archaea as Haloferax and Thermococcus are here described as EPS producers underlining biopolymer hyperproduction, related fermentation strategies including the effects of the chemical composition of the media, the physical parameters of the growth conditions and the genetic and predicted experimental design tools.

New regulatory mechanisms in the biosynthesis of pheomelanins: rearrangement vs. redox exchange reaction routes of a transient 2H-1,4-benzothiazine-o-quinonimine intermediate

Pheomelanins, the typical epidermal pigments of red haired, Celtic-type Caucasians, arise from oxidative cyclization of cysteinyldopas, mainly the 5-S-isomer CD, via 1,4-benzothiazines. However, the mechanism and the relative yields of formation of these intermediates have remained poorly defined. We have now examined the course of the oxidation of CD at physiological pHs, under different reaction conditions. Surprisingly, a consumption of CD far exceeding the stoichiometry of the oxidant was observed at low oxidant-to-substrate ratios, low temperatures and high substrate concentrations. The yields of the 3,4-dihydro-1,4-benzothiazine-3-carboxylic acid DHBCA vs. the non-carboxylated analogue DHB in the oxidation mixture, after NaBH4 reduction, were also found to depend markedly on the reaction conditions. Based on these and other results, a reaction scheme is proposed involving a transient o-quinonimine generated by oxidative cyclization of CD to which three different paths are offered, namely redox exchange with CD to give DHBCA (path A) or intramolecular rearrangement with (path B) or without (path C) decarboxylation, leading to the benzothiazine BTZ and the 3-carboxy analogue BTZCA, respectively. The relative operation of path A vs. path C was assessed by deuterium labeling experiments. These findings point to new mechanisms of regulation of the initial steps of pheomelanogenesis, bearing significant implications on the structure of the final pigment.

Transient quinonimines and 1,4-benzothiazines of pheomelanogenesis: new pulse radiolytic and spectrophotometric evidence.

Biosynthetic and model in vitro studies have shown that pheomelanins, the distinctive pigments of red human hair, arise by oxidative cyclization of cysteinyldopas mainly 5-S-cysteinyldopa (1) via a critical o-quinonimine intermediate, which rearranges to unstable 1,4-benzothiazines. To get new evidence for these labile species, fast time resolution pulse radiolytic oxidation by dibromide radical anion of a suitable precursor, the dihydro-1,4-benzothiazine-3-carboxylic acid 7 was performed in comparison with that of 1. In the case of 7, dibromide radical anion oxidation leads over a few microseconds (k = 2.1 x 10(9) M(-1) s(-1)) to a phenoxyl radical (lambda(max) 330 nm, epsilon = 6300 M(-1) cm(-1)) which within tens of milliseconds gives rise with second-order kinetics (2k = 2.7 x 10(7) M(-1) s(-1)) to a species exhibiting an absorption maximum at 540 nm (epsilon = 2200 M(-1) cm(-1)). This was formulated as the o-quinonimine 3 arising from disproportionation of the initial radical. The quinonimine chromophore is converted over hundreds of milliseconds (k = 6.0 s(-1)) to a broad maximum at around 330 nm interpreted as due to a 1,4-benzothiazine or a mixture of 1,4-benzothiazines, which as expected are unstable and subsequently decay over a few seconds (k = 0.5 s(-1)). Interestingly, the quinonimine is observed as a labile intermediate also in the alternative reaction route examined, involving cyclization of the o-quinone (lambda(max) 390 nm, epsilon = 6900 M(-1) cm(-1)) arising by disproportionation (2k = 1.7 x 10(8) M(-1) s(-1)) of an o-semiquinone (lambda(max) 320 nm, epsilon = 4700 M(-1) cm(-1)) directly generated by dibromide radical anion oxidation of 1. Structural formulation of the 540 nm species as an o-quinonimine was further supported by rapid scanning diode array spectrophotometric monitoring of the ferricyanide oxidation of a series of model dihydrobenzothiazines.

Heavy metal resistance of some thermophiles : potential use of α-amylase from Anoxybacillus amylolyticus as a microbial enzymatic bioassay

Six thermophilic extremophiles, Anoxybacillus amylolyticus, Geobacillus thermoleovorans, Geobacillus thermoleovorans subspecies stromboliensis, Geobacillus toebii subspecies decanicus, Bacillus thermantarcticus and Thermus oshimai, isolated from different environmental sites, were studied for their heavy metal resistance. The effects of heavy metals on microorganism growth were studied here in a pilot fermenter tank spiked with various trace metals, (Ni(2+), Zn(2+), Co(2+), Hg(2+), Mn(2+), Cr(6+), Cu(2+), Fe(3+) and Cd(2+)) at concentrations spanning from 0.01 to 20 mM. Trace metal toxicity varied depending on the species and metal considered. Among the tested microorganisms, attention was focused on alpha-amylase producing-A. amylolyticus, an acidothermophilic bacterium recently isolated from geothermal soil samples from Mount Rittmann in Antarctica. The effect of heavy metals on the biosynthesis and activity of alpha-amylase of A. amylolyticus was investigated. When bacteria were grown in the presence of heavy metals, a decrease in alpha-amylase activity, correlated with a decrease in alpha-amylase production, was observed, suggesting an effect on the biosynthesis of the enzyme. A decrease in enzyme activity was also noted when the assay was performed in the presence of heavy metals. Thus, alpha-amylase could represent a potential sensitive bioassay for detecting trace heavy metals.

Polysaccharides from Wastes of Vegetable Industrial Processing: New Opportunities for Their Eco-Friendly Re-Use

Polysaccharides are the most abundant natural biopolymers and represent by far the largest group of polymers produced in the world: indeed more than 150,000 M tons of polysaccharides per year are produced in comparison with about 140 M tons of synthetic polymers (Navard, 2005). Polysaccharides are widely diffused in nature: they can be found in plants, animals and microorganisms, performing different fundamental biological functions. Depending on their functional role, they can be distinguished in: energy reserve substances (e.g. starch or inulin in plants, glycogen in animals), structural elements maintaining mechanical shape and rigidity of the living cells (cellulose, hemicellulose and pectin in plant cell wall, chitin in arthropod exoskeletons), and water-binding elements (agar, pectin and alginates in plants, mucopolysaccharides in animals). About 99% of total natural polysaccharides are located in plants and vegetables that consequently represent a major renewable source of these biopolymers exploitable for different purposes. Indeed, polysaccharides are presently used in all sectors of human activities and in several application such as: food nutrients, food additives and feed production; material science concerning the formulation of polymeric materials for different biotechnological applications; health care for biocompatible materials, drug delivery or as source of biologically active molecules; sustainable energy production by means of biofuels generation (Persin et al., 2010). The main and most abundant polysaccharides from plants and vegetables include starch, cellulose and hemicelluloses, inulin and pectins that, thanks to their useful physicalchemical properties such as emulsifying power, viscoelasticity, polyelectrolyte conformation, adherence, bio-compatibility, stabilizing power, et cetera are usually exploited in the above mentioned fields of application. In addition, several other polysaccharides with different structures and properties are continuously isolated and are under investigation in relation to their features and potential employment in several industrial sectors.

The hemicellulose extract from Cynara cardunculus: a source of value-added biomolecules produced by xylanolytic thermozymes

The Cynara cardunculus hemicellulose fraction was recovered from its stem and leaf biomass and converted into valuable molecules by exploiting the extracellular xylanase and β-xylosidase activities produced by the thermophilic bacterium Geobacillus thermantarcticus. Several degradation procedures for the arabinoglucuronoxylan extract were proposed by using efficient and different enzymatic preparations, containing both or only one xylanolytic activity of G. thermantarcticus. In particular, when the xylanase and β-xylosidase activities were used separately in hydrolysis reactions, xyloglucurono-oligosaccharides or xylose were obtained with a yield of 32% and 62.6% respectively, with reference to the hemicellulosic extract. Furthermore, the synergic action of β-xylosidase/xylanase activities was exploited in transglycosylation processes for the production of xylo-conjugated compounds; xylosides of primary alcohols with increasing carbon chains and aromatic alcohols were produced starting from the C. cardunculus hemicellulose, which was selected as a cheap donor. When 2-phenoxyethanol was selected as an acceptor, 2-phenoxyethyl β-xyloside, xylobioside and xylotrioside were prepared with a yield of 38.5% with respect to the hemicellulosic extract and spectroscopically characterized.

Nesterenkonia aurantiaca, sp. nov., an alkaliphilic actinobacterium isolated from Cape King (Antarctica).

A Gram-stain-positive, non-endospore-forming, haloalkaliphilic actinobacterium, strain CK5T, was isolated from a soil sample, collected at Cape King (Antarctica), and its taxonomic position was investigated by using a polyphasic approach. Cells were cocci with orange pigmentation, non-motile and grew optimally at 25 °C and pH 9.0-9.5 in the presence of 2 % (w/v) NaCl. Cellular membrane contained MK-7 (72 %) and MK-8 (28 %), and anteiso-C15 : 0 (64.8 %), iso-C16 : 0 (13.3 %), n-C17 : 0 (9.9 %), n-C16 : 0 (4.0 %), n-C14 : 0 (3.7 %) as major cellular fatty acids. The DNA G+C content was 64.8 mol%. Strain CK5T, based on the 16S rRNA gene sequence similarity, was most closely related to Nesterenkonia jeotgali JG-241T (99.5 %), Nesterenkonia sandarakina YIM 70009T (99.4 %), Nesterenkonia lutea YIM 70081T (99.4 %), Nesterenkonia halotolerans YIM 70084T (99.3 %), Nesterenkonia xinjiangensis YIM 70097T (97.2 %), Nesterenkonia flava CAAS 251T (97.1 %) and Nesterekonia aethiopica CCUG 48939T (97.1 %). Strain CK5T revealed 31 % DNA-DNA relatedness with respect to N. sandarakina DSM 15664T, 29 % with respect to N. jeotgali DSM 19081T, 10 % with respect to N. lutea DSM 15666T and 1 % with respect to N. halotolerans, DSM 15474T, N. xinjiangensis DSM 15475T, N. aethiopica DSM 17733T and N. flava DSM 19422T. On the basis of 16S rRNA gene sequences, DNA-DNA hybridization and chemotaxonomic characteristics, strain CK5T represents a novel species of the genus Nesterenkonia, for which the name Nesterenkonia aurantiaca sp. nov. is proposed. The type strain is CK5T ( = DSM 27373T = JCM 19723T).

Degradative actions of microbial xylanolytic activities on hemicelluloses from rhizome of Arundo donax.

Polysaccharidases from extremophiles are remarkable for specific action, resistance to different reaction conditions and other biotechnologically interesting features. In this article the action of crude extracts of thermophilic microorganisms (Thermotoga neapolitana, Geobacillus thermantarcticus and Thermoanaerobacterium thermostercoris) is studied using as substrate hemicellulose from one of the most interesting biomass crops, the giant reed (Arundo donax L.). This biomass can be cultivated without competition and a huge amount of rhizomes remains in the soil at the end of cropping cycle (10–15 years) representing a further source of useful molecules. Optimization of the procedure for preparation of the hemicellulose fraction from rhizomes of Arundo donax, is studied. Polysaccharidases from crude extracts of thermophilic microorganisms revealed to be suitable for total degradative action and/or production of small useful oligosaccharides from hemicelluloses from A. donax. Xylobiose and interesting tetra- and pentasaccharide are obtained by enzymatic action in different conditions. Convenient amount of raw material was processed per mg of crude enzymes. Raw hemicelluloses and pretreated material show antioxidant activity unlike isolated tetra- and pentasaccharide. The body of results suggest that rhizomes represent a useful raw material for the production of valuable industrial products, thus allowing to increase the economic efficiency of A. donax cultivation.