Purification and Characterization of Anabaena flos-aquae Phenylalanine Ammonia-Lyase as a Novel Approach for Myristicin Biotransformation.

Phenylalanine ammonia-lyase (PAL) catalyzes the reversible deamination of phenylalanine to cinnamic acid and ammonia. Algae have been considered as biofactories for PAL production, however, biochemical characterization of PAL and its potency for myristicin biotransformation into MMDA (3-methoxy-4, 5-methylenedioxyamphetamine) has not been studied yet. Thus, PAL from Anabaena flos-aquae and Spirulina platensis has been purified, comparatively characterized and its affinity to transform myristicin was assessed. The specific activity of purified PAL from S. platensis (73.9 µmol/mg/min) and A. flos-aquae (30.5 µmol/mg/min) was increased by about 2.9 and 2.4 folds by gel-filtration comparing to their corresponding crude enzymes. Under denaturing-PAGE, a single proteineous band with a molecular mass of 64 kDa appeared for A. flos-aquae and S. platensis PAL. The biochemical properties of the purified PAL from both algal isolates were determined comparatively. The optimum temperature of S. platensis and A. flos-aquae PAL for forward or reverse activity was reported at 30°C, while the optimum pH for PAL enzyme isolated from A. flos-aquae was 8.9 for forward and reverse activities, and S. platensis PAL had maximum activities at pH 8.9 and 8 for forward and reverse reactions, respectively. Luckily, the purified PALs have the affinity to hydroaminate the myristicin to MMDA successfully in one step. Furthermore, a successful method for synthesis of MMDA from myristicin in two steps was also established. Gas chromatography-mass spectrometry (GC-MS) analysis was conducted to track the product formation.

Effects of nutrient loading on Anabaena flos-aquae biofilm: biofilm growth and nutrient removals.

Effects of three different nutrient loadings (low nutrient loading, medium nutrient loading and high nutrient loading, denoted as LNS, MNS and HNS, respectively) on the structure and functions of algal biofilm using Anabaena flos-aquae were investigated using synthetic wastewater. Nutrients removal efficiencies, biofilm thickness, microalgae dehydrogenase activity (DHA) and exopolysaccharide (EPS) productions were examined. Results showed that the changes of nutrient concentration were insignificant after 4 days of experiment for the case of HNS condition; 9 days for the case of MNS condition, and 6 days for the case of LNS condition, respectively. The biofilm thickness, nutrient removal efficiencies, algae DHA and EPS productions increased with the increase of nutrient loadings in synthetic wastewater. For the case of HNS condition, the microalgal biofilm exhibited the best performance in terms of C, N and P removal efficiencies, reaching the removal rates of 68.45, 3.56 and 1.61 mg·L(-1)·d(-1) for C, N, P, respectively. This was likely because, fact with the high nutrient loading, the high biological activity could be achieved, thus resulting in high nutrient removals. The thickness of the biofilm in HNS condition was 75 µm, which was closely related to EPS production. DHA and EPS concentrations were 7.24 and 1.8 × 10(-2) mg·mm(-2), respectively. It was also shown that apart from the nutrient loading, the structure and functions of microalgal biofilm were also influenced by other factors, such as illumination and temperature.

Ecotoxicological studies of micro- and nanosized barium titanate on aquatic photosynthetic microorganisms.

The interaction between live organisms and micro- or nanosized materials has become a current focus in toxicology. As nanosized barium titanate has gained momentum lately in the medical field, the aims of the present work are: (i) to assess BT toxicity and its mechanisms on the aquatic environment, using two photosynthetic organisms (Anabaena flos-aquae, a colonial cyanobacteria, and Euglena gracilis, a flagellated euglenoid); (ii) to study and correlate the physicochemical properties of BT with its toxic profile; (iii) to compare the BT behavior (and Ba(2+) released ions) and the toxic profile in synthetic (Bold's Basal, BB, or Mineral Medium, MM) and natural culture media (Seine River Water, SRW); and (iv) to address whether size (micro, BT MP, or nano, BT NP) is an issue in BT particles toxicity. Responses such as growth inhibition, cell viability, superoxide dismutase (SOD) activity, adenosine-5-triphosphate (ATP) content and photosynthetic efficiency were evaluated. The main conclusions are: (i) BT have statistically significant toxic effects on E. gracilis growth and viability even in small concentrations (1µgmL(-1)), for both media and since the first 24 h; on the contrary of on A. flos-aquae, to whom the effects were noticeable only for the higher concentrations (after 96 h: ≥75 µg mL(-1) for BT NP and =100 µg mL(-1) for BT MP, in BB; and ≥75 µg mL(-1) for both materials in SRW), in spite of the viability being affected in all concentrations; (ii) the BT behaviors in synthetic and natural culture media were slightly different, being the toxic effects more pronounced when grown in SRW - in this case, a worse physiological state of the organisms in SRW can occur and account for the lower resistance, probably linked to a paucity of nutrients or even a synergistic effect with a contaminant from the river; and (iii) the effects seem to be mediated by induced stress without a direct contact in A. flos-aquae and by direct endocytosis in E. gracilis, but in both organisms the contact with both BT MP and BT NP increased SOD activity and decreased photosynthetic efficiency and intracellular ATP content; and (iv) size does not seem to be an issue in BT particles toxicity since micro- and nano-particles produced significant toxic for the model-organisms.

Effects of monosulfuron on growth, photosynthesis, and nitrogenase activity of three nitrogen-fixing cyanobacteria.

Application of monosulfuron, a new sulfonylurea herbicide, produced a simulative effect on heterocyst formation and nitrogenase activity but an inhibitory effect on photosynthesis, i.e., a lower net photosynthetic rate, fewer photosynthetic pigments, and a smaller Fv/Fm ratio at increasingly higher monosulfuron concentrations (0.001-10 mg/l) for three nonspecific filamentous nitrogen-fixing cyanobacteria: Anabaena azollae, A. flos-aquae, and A. azotica. The decrease in biliprotein of algal cells was less than that of carotenoid and chlorophyll-a. Monosulfuron was more readily degraded and less accumulated in A. azotica compared with A. azollae and A. flos-aquae. The three algae exhibited varying degrees of sensitivity to monosulfuron: Calculated 50% inhibition concentrations (IC(50)s) of algal growth and no observed-effect concentration (NOEC) values after 4 days of treatment were 0.014 and 0.005, 0.029 and 0.019, and 0.22 and 0.075 mg/l for A. flos-aquae, A. azollae, and A. azotica, respectively. Normal agricultural use of monosulfuron at postemergence rates of 0.3-0.8 mg/l in rice fields will likely be toxic to these three ubiquitous nitrogen-fixing cyanobacteria. Low-dose monosulfuron application (<0.1 mg/l) enables growth of the more tolerant A. azotica as biofertilizer, and the use of photosynthetic efficiency and growth rates as sensitive-indicator indexes of toxicity to nitrogen-fixing cyanobacteria are recommended.

A correlation matrix of alkaline phosphatase and sporulation in diazotrophic cyanobacteria and its thermo-tolerant mutant.

Alkaline phosphatase (phosphomonoesterase i.e. PMEase) activity in heterocystous cyanobacteria Anabaena flos-aquae, Nostoc calcicola, Calothrix brevissima, Scytonema javanicum and Hapalosiphon intricatus is known to be temperature and pH dependent. Maximum level of enzyme activity was recorded at either 35 degrees C or 37.5 degrees C. Also, the cell bound phosphomonoesterase enzyme was shown to exhibit pH optima of 10.0 or 10.2. A thermo-tolerant (tr) mutant isolated after MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis in Calothrix brevissima exhibited 10 degrees C higher temperature optima and comparatively high pH optima (pH 10.4) for phosphomonoesterase enzyme. The mutant grew with a maximum growth rate (k) at 50 degrees C. Activation energy (Ea) for cyanobacterial strains was in a narrow range between 45 to 52 kJ mol(-1). A little variation in temperature and pH optima was also observed in phosphomonoesterase activity of Calothrix brevissima and its thermo-tolerant mutant while utilizing various organic phosphates as substrate what indicated the substrate dependence temperature and pH optima. Cyanobacterial strains grown at their respective temperature and pH optima differentiated spores less frequently though, coupled with early initiation of spore.