Free volume variations of Nylon12/Polyvinyl alcohol (Nylon12/PVA) blends treated in supercritical carbon dioxide (ScCO₂) were investigated using positron annihilation lifetime spectroscopy. It is found that o-Ps lifetime τ₃ and the corresponding intensity I₃ in Nylon12/PVA decrease with increase of the PVA content. Being treated in ScCO₂ at 50°C and 20 MPa for 1 h, the free volume holes in Nylon12/PVA blends expand to different extents depending on the weight ratio of PVA. After releasing of CO₂, the free volume size decreases as a function of the elapsed time due to the molecular relaxation, and the relaxation time constant depends on the content of PVA in the blends.
His296 of Zymomonas mobilis levansucrase (EC 2.4.1.10) is crucial for the catalysis of the transfructosylation reaction. The three-dimensional structures of levansucrases revealed the His296 is involved in the substrate recognition and binding. In this study, nine mutants were created by site-directed mutagenesis, in which His296 was substituted with amino acids of different polarity, charge and length. The substitutions of His296 with Arg or Trp retained partial hydrolysis and transfructosylation activities. The positively charged Lys substitution resulted in a 2.5-fold increase of sucrose hydrolysis. Substitutions with short (Cys or Ser), negatively charged (Glu) or polar (Tyr) amino acids virtually abolished both the activities. Analysis of transfructosylation products indicated that the mutants synthesized different oligosaccharides, suggesting that amino acid substitutions of His296 strongly affected both the enzyme activity and transfructosylation products.
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