Increasing the thermostability of d-xylose isomerase by introduction of a proline into the turn of a random coil

Abstract

Thermostability can be increased by introducing prolines at suitable sites in target proteins. Two single (G138P, G247D) mutants and one double (G138P/G247D) mutant of xylose isomerase from Streptomyces diastaticus No.7, strain M1033 have been constructed by site-directed mutagenesis. With respect to the wild-type enzyme, G138P showed about a 100% increase in thermostability, and G247D showed an increased catalytic activity. Significantly, the double mutant, G138P/G247D displayed even higher activity than G247D and better heat stability than G138P. Its half life was about 2.5-fold greater than the wild-type enzyme, using xylose as a substrate. Molecular modelling suggested that the introduction of a proline residue in the turn of a random coil may cause the surrounding conformation to be tightened by reducing the backbone flexibility. The change in thermostability can, therefore, be explained based on changes in the molecular rigidity. Furthermore, the improvements in the properties of the double mutant indicated that the advantages of two single mutants can be combined effectively.

Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

“…Introduction of proline residues at certain positions has been used successfully to improve the thermostabilities of many enzymes (39)(40)(41)(42)(43)(44). In the present study, we introduced proline at sites 81 and 185, which are located at the second position of the ␤-turn and the N-cap of the ␣-helix (Fig.…”

Mesophile versus Thermophile: Insights Into the Structural Mechanisms of Kinetic Stability

“…Indeed, proline residues have been shown to increase thermostability in other systems, [36][37][38] especially when found in turn positions. [39][40][41][42] Intriguingly, the only related protease to show significant sequence homology to TFPA in this loop (including a proline at residue 67) is also from an extreme thermophile, Pyroccocus furiosus (data not shown).…”

Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

“…The thermostability of some enzymes was increased by the proline substitution, such as xylose isomerase [15], mesophilic alcohol dehydrogenase [13], ␣-glucosidase [16] and glycosidase [17]. The substitution of proline residues at selected positions might be a potential feasible strategy to improve the thermostability or catalytic efficiency of CsCE.…”