Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei

Abstract

Xylanases isolated from microorganisms such as the Trichoderma reesei have attracted considerable research interest because of their potential in various industrial applications. However, naturally isolated xylanases cannot withstand harsh conditions such as high temperature and basic pH. In this study, we performed structural analysis of the major T. reesei xylanase (Xyn2), and novel flexible regions of the enzyme were identified based on B-factor, a molecular dynamics (MD) parameter. To improve thermostability of the Xyn2, disulfide bonds were introduced into the unstable flexible region by using site-directed mutagenesis and two recombinant xylanases, XM1 (Xyn2Cys12-52) and XM2 (Xyn2Cys59-149) were successfully expressed in Pichia pastoris. Secreted recombinant Xyn2 was estimated by SDS-PAGE to be 24 kDa. Interestingly, the half-lives of XM1 and XM2 at 60°C were 2.5- and 1.8- fold higher, respectively than those of native Xyn2. The XM1 also exhibited improved pH stability and maintained more than 60% activity over pH values ranging from 2.0 to 10.0. However, the specific activity and catalytic efficiency of XM1 was decreased as compared to those of XM2 and native Xyn2. Our results will assist not only in elucidating of the interactions between protein structure and function, but also in rational target selection for improving the thermostability of enzymes.

Fig 1. Structural analysis of T. reesei Xyn2 and molecular design.

(A)Visualization of unstable regions of Xyn2; (B) Locations of the predicted disulfide bond in Xyn2.

Fig 2. SDS-PAGE analysis of Xyn2 and its mutants.

M: Mark; Lanes 1, 3, 5: Expression product of xyn2, XM1 and XM2; Lanes 2, 4, 6: Expression product of xyn2, XM1 and XM2 treated with Endo H_f.

Fig 3. Influence of glycosylation on the enzymatic activity of Xyn2 and mutated enzymes.

Control: untreated enzymes; Deglycosylation: enzymes treated with endo H_f.

Fig 4. Validation of disulfide bonds in XM1 and XM2.

(A) SDS-PAGE. M: Marker; Lane 1, 2, and 3: the native Xyn2 were treated with 0, 2.5, and 10 mmol DDT; Lane 4: XM1; Lane 5: XM1 treated with 2.5 nmol DDT; Lane 6: XM1 treated with 10 nmol DDT; Lane 7: XM2; Lane 8: XM2 treated with 2.5 mmol DDT; Lane 9: XM2 treated with 10 mmol DDT. (B) Influence of amino acid mutation on enzymatic activity.

Fig 5. Enzymatic properties of the Xyn2 and mutated enzymes.

(A) Influence of temperature on the enzyme activity; (B) The thermostability of the enzymes; (C) Influence of pH on the enzyme activity; (D) The pH stability of the enzymes.