| Summary: | Dissertação de mestrado em Genética Molecular Xylan is the main carbohydrate of the hemicellulosic fraction of plant tissues and accounts for one third of all renewable organic carbon available. Xylanases (EC 3.2.1.8), act in nature by catalysing the hydrolysis of xylan molecules into oligomeric and monomeric pentosan units that bacteria and fungi can use as a carbon source. Xylanase producers have been isolated from all ecological niches where plant material accumulates. pXyl, a highly active psychrophilic xylanase, has previously been isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a. This cold-adapted xylanase, retains 90 % and 60 % of its activity at 20 ºC and 5 ºC, respectively. It is fully stable (at 25 ºC) between pH 6 and 9 and has maximum activity (>80 % at 25 ºC) between pH 5 and 7.8. At lower pHs irreversible inactivation, protein precipitation and eventually protein unfolding occurs. To study the influence of surface hydrophobic interactions on low pH adaptation and protein precipitation, site-directed mutagenesis and a comparative characterisation of the pH dependency of activity, stability and precipitation of mutant and wild-type enzymes was carried out. In particular, surface hydrophobic residues in an exposed patch as well as neighbouring negative residues were mutated to the polar amino acids serine and/or lysine. Of the 10 mutants prepared, Y43S, V58S and E94S revealed improvements in acid pH stability as compared to the wild-type enzyme, with reduced irreversible inactivation, a maintenance of tertiary structure as monitored by tryptophan fluorescence and increases in the solubility at pH 5 and 5.5 being observed. Y43S and E94S also increased their melting temperature at pH 5 by 2.0 and 0.9 ºC, respectively, whereas for V58S this was decreased 2.2 ºC. It is suggested that specific exposed surface hydrophobes as well as negative residues which display an increased hydrophobicity on protonation at low pHs lead to the increased precipitation of pXyl at acidic pHs. ...
|
|---|