Engineering the thermostability of Trichoderma reesei endo-1,4-β-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 N22D, N38E, and Q162H. With a half-life of similar to56 h at 65degreesC. the thermostability of this sevenfold Mutant was similar to5,000 times higher than that of TRX II, and the half-life was 25 min even at 75degreesC. The thermostability of this mutant was similar to30 times higher than that of the corresponding mutant missing the bridge between positions 2 and 28. The extensive stabilization Lit two protein regions did not after the kinetic properties of the sevenfold mutant front that of the wild-type TRX II. The combination of disulphide bridges enhanced significantly the pH-dependent stability in a wide pH range.