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.
Xylose isomerase produced by Bacillus thermoantarcticus was purified 73-fold to homogeneity and its biochemical properties were determined. It was a homotetramer with a native molecular mass of 200 kDa and a subunit molecular mass of 47 kDa, with an isoelectric point at 4.8. The enzyme had a K(m) of 33 mM for xylose and also accepted D-glucose as substrate. Arrhenius plots of the enzyme activity of xylose isomerase were linear up to a temperature of 85 degrees C. Its optimum pH was around 7.0, and it had 80% of its maximum activity at pH 6.0. This enzyme required divalent cations for its activity and thermal stability. Mn(2+), Co(2+) or Mg(2+) were of comparable efficiency for xylose isomerase reaction, while Mg(2+) was necessary for glucose isomerase reaction.
A high thermostable extracellular protease was purified to homogeneity and characterised from Bacillus thermantarcticus, strain M1. The molecular mass was about 42 kDa. Almost total inhibition of protease by phenyl methyl sulphonylfluoride (PMSF), suggested that the enzyme belonged to the serine protease family. The enzyme was active and stable in a broad range of pH with an optimum at pH 7.0. The protease showed the highest activity at 70 °C and was stable for 24 h at 70 °C, with an increase of the enzymatic activity of about 4 times, in the presence of CaCl 2 . The protease retained about 50% activity after 3 h of incubation in the presence of CaCl 2 with various commercial detergents. Purified protease was found to be stable, for one week, in presence of DMSO, methanol, ethanol, acetonitrile, isopropanol.
In this study we suggest a simplified and effective method to directly recover polyhydroxyalkanoates (PHAs) from humid biomass of Halomonas campaniensis with no pre-treatment steps. Sodium dodecyl sulphate (SDS) was directly added to dispersed biomass of cultured micro-organism (w/w ratio: 1) in distilled water followed by shaking, heat treatment, and washing steps. The purity of the recovered PHAs synthesized by H. campaniensis was over 95%, regardless of the cell concentrations and the best yield was 12% (w/w) of the cell wet weight when the micro-organism was cultivated in a glucose-based medium or a glucose/propionate-based medium. MS spectroscopy and 1 H, 13 C-NMR analysis were used to chemically characterize the PHAs; their thermal characteristics were obtained using a differential scanning calorimeter and the average viscosity molecular weight was assessed through specific viscosity measurements. Due to its ease and velocity, our simplified method is suitable for the detection and recovery of PHAs from humid biomasses with high yield and purity. The method, which is quick and at low environmental impact, is very valuable for the simultaneous testing of cultures grown with different inducers for PHAs having particular chemical/physical characteristics.
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