Thermostability can be increased by introducing prolines at suitable sites in target
proteins. In this study, we compared five thermostable alpha-glucosidases and the
moderate thermostable alpha-glucosidase (TtGluA) from Thermoanaerobacter tengcongensis
MB4. Based on the amino acid sequence alignment, four sites (Leu152, Asn208, Lys285,
and Thr430) of TtGluA were chosen for proline substitution to improve its thermostability.
Thermostability of mutants L152P, K285P, and T430P increased evidently, but no thermostability
improvement was observed for N208P. Compared to the wild-type enzyme, T(50)(15) of
T430P had a rise of 2 degrees C without distinct loss of activity. However, T(50)(15)
values of L152P and K285P increased 2 degrees C and 10.5 degrees C, respectively,
while retaining activity of only 26.6% and 24.9% of wild-type enzyme. The K(m) of
L152P, K285P, T430P and wild-type enzyme was 1.61, 0.32, 1.64, and 1.08 mM, respectively.
These indicate that the selected sites are not only important for the thermostability
but also related to the substrate binding and catalytic activity of TtGluA. The CD
spectra analysis of the improved mutants and wild-type enzyme showed no distinct changes
in their secondary structures. Combining analysis of secondary structure prediction
and 3D structure modeling, the proline substitution at the three sites stabilized
TtGluA possibly by reducing the flexibility of loop and random coil or (and) increasing
the hydrophobic effect at these strategic regions with no evident structure change.
2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.