Engineering the thermostability of Trichoderma reesei endo-1,4-beta-xylanase II by combination of disulphide bridges.
Disulphide bridges were introduced in different combinations into the N-terminal region and the single alpha-helix of mesophilic Trichoderma reesei xylanase II (TRX II). We used earlier disulphide-bridge data and designed new disulphide bridges for the combination mutants. The most stable mutant contained two disulphide bridges (between positions 2 and 28 and between positions 110 and 154, respectively) and the mutations N11D, N38E, and Q162H. With a half-life of approximately 56 h at 65 degrees C, the thermostability of this sevenfold mutant was approximately 5,000 times higher than that of TRX II, and the half-life was 25 min even at 75 degrees C. The thermostability of this mutant was approximately 30 times higher than that of the corresponding mutant missing the bridge between positions 2 and 28. The extensive stabilization at two protein regions did not alter the kinetic properties of the sevenfold mutant from that of the wild-type TRX II. The combination of disulphide bridges enhanced significantly the pH-dependent stability in a wide pH range.
Laboratory of Bioprocess Engineering, Helsinki University of Technology, P.O. Box 6100, 02015-HUT, Helsinki, Finland.
Hairong
H
Xiong
Fred
F
Fenel
Matti
M
Leisola
Ossi
O
Turunen
D004220:Disulfides / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D013696:Temperature; D013997:Time Factors; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D004220:Disulfides; D043364:Endo-1,4-beta Xylanases