Taka-amylase A (TAA) proteins purified from Aspergillus oryzae were investigated using dynamic and static light scattering for solutions containing poly(ethylene)glycol (PEG) with a molecular weight of 8000 as an association-inducing reagent. The hydrodynamic TAA monomer radius at a zero protein concentration gradually decreased from 3.2 to 2.0 nm with an increase in the PEG concentration from 0 to 12.5% (w/v). The molecular weight of TAA monomers at 0% PEG, 49.0 kDa, decreased to 31.0 kDa at 12.5% PEG. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis analysis showed that the monomer-band position decreased with an increase in the PEG concentration. While these findings suggest PEG-induced fragmentation of monomers, circular dichroism measurements showed no difference between 12.5 and 0% PEG solutions in the secondary structure of TAA monomers. Analytical ultracentrifugation with a 12.5% PEG solution gave a TAA molecular weight of 51.0 kDa, almost equal to that for a 0% PEG solution measured using static light scattering. These results suggest that a PEG-induced network locally formed in the solution and affected the dynamics and the scattering intensity of the TAA monomers. The existence of such a solution structure makes it impossible to explain the behavior of monomers on the basis of simple attractive depletion interaction by PEG.

Crystallization of enzymes in presence of impurities is important for clarifying the role of enzymes in natural world. Although it is proposed that impurities inhibit nucleation of enzyme crystallization, details are unclear. In this study, crystallization of cellobiohydrolase from Aspergillus niger was investigated by dynamic and time-resolved static light scattering using cellobiose as an impurity. We aimed to clarify how cellobiose inhibits cellobiohydrolase crystallization and to crystallize cellobiohydrolase in concentrated cellobiose without using seeds. The contribution of attractive forces to total intermolecular interactions of cellobiohydrolase monomers increased with the molar ratio of cellobiose/cellobiohydrolase (R(cb/ce)). Association dynamics of cellobiohydrolase using lithium sulfate, however, showed that the initial aggregation rate decreased with an increase in R(cb/ce). Because binding sites of cellobioses to cellobiohydrolase molecules differed from those for the growth of protein crystals, the binding of cellobioses would increase the chemical potential of the cellobiohydrolase monomers, which gradually reduced supersaturation for growth as the aggregate size increased. This result was in contrast with the conventional idea that cellobiose inhibits the nucleation of cellobiohydrolase crystals. Gentle agitation of cellobiose-containing cellobiohydrolase solutions during sitting-drop vapor-diffusion growth resulted in the growth of cellobiohydrolase single crystals for all R(cb/ce) conditions without using seeds.

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