Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling.
The thermostability of Streptomyces lividans xylanase B (SlxB-cat) was significantly increased by the replacement of its N-terminal region with the corresponding region from Thermomonospora fusca xylanase A (TfxA-cat) without observing a decrease in enzyme activity. In spite of the significant similarity between the amino acid sequences of the two xylanases, their thermostabilities are quite different. To facilitate an understanding of the contribution of structure to the thermostability observed, chimaeric enzymes were constructed by random gene shuffling and the thermostable chimaeric enzymes were selected for further study. A comparative study of the chimaeric and parental enzymes indicated that the N-terminus of TfxA-cat contributed to the observed thermostability. However, too many substitutions decreased both the thermostability and the activity of the enzyme. The mutants with the most desirable characteristics, Stx15 and Stx18, exhibited significant thermostabilities at 70 degrees C with optimum temperatures which were 20 degrees C higher than that of SlxB-cat and equal to that of TfxA-cat. The ability of these two chimaeric enzymes to produce reducing sugar from xylan was enhanced in comparison with the parental enzymes. These results suggest that these chimaeric enzymes inherit both their thermostability from TfxA-cat and their increased reactivity from SlxB-cat. Our study also demonstrates that random shuffling between a mesophilic enzyme and its thermophilic counterpart represents a facile approach for the improvement of the thermostability of a mesophilic enzyme.
Shibuya
H
H
Wood Chemistry Division, Forestry and Forest Products Research Institute, Kukizaki, Ibaraki, 305-8687, Japan. bushi@ffpri.affrc.go.jp
Kaneko
S
S
Hayashi
K
K
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000096:biosynthesis; D004795:Enzyme Stability; D015321:Gene Rearrangement / Q000502:physiology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism*
D002241:Carbohydrates; D011993:Recombinant Fusion Proteins; D014990:Xylans; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase