A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-β-xylanase II

We have successfully engineered a disulphide bridge into the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII) by substituting Thr-2 and Thr-28 with cysteine. The T2C:T28C mutational changes increased the half-life in thermal inactivation of this mesophilic enzyme from similar to40 s to similar to20 min at 65degreesC, and from less than 10 s to similar to6 min at 70degreesC. Therefore, the N-terminal disulphide bridge enables the use of XYNII at substantially higher temperatures than permitted by its native mesophilic counterpart. Altogether, thermostability increased by about 15degreesC. The kinetic properties of the mutant XYNII were maintained at the level of the wild type enzyme. Our findings demonstrated that a properly designed disulphide bridge, here within the N-terminal region of XYNII, can be very effective in resisting thermal inactivation. (C) 2003 Elsevier B.V. All rights reserved.