Enhancing the thermostability of alpha-glucosidase from Thermoanaerobacter tengcongensis MB4 by single proline substitution.
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.
Zhou
Cheng
C
State Key Lab of Microbial Resources, Institute of Microbiology, Chinese Academy of Science, Beijing 100101, China.
Xue
Yanfen
Y
Ma
Yanhe
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006358:Hot Temperature; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011392:Proline / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D046968:Thermoanaerobacter / Q000235:genetics* / Q000378:metabolism*; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011392:Proline; D000520:alpha-Glucosidases