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.