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.
Highlights
Xylan is a major component of the plant cell wall consisting of a β-D-1,4-linked xylopyranoside backbone substituted with acetyl, arabinosyl, and glucuronosyl side chains [1, 2]
We successfully introduced disulfide bonds into the unstable flexible regions of T. reesei xylanase 2 (Xyn2) by using site-directed mutagenesis, and two mutated xylanases were successfully expressed in Pichia pastoris
Introduction of the disulfide bonds resulted in elevated thermostability and pH stability in the T. reesei Xyn2
Summary
Xylan is a major component of the plant cell wall consisting of a β-D-1,4-linked xylopyranoside backbone substituted with acetyl, arabinosyl, and glucuronosyl side chains [1, 2]. Hydrolysis of the xylan backbone is catalysed by endo-1,4-β-xylanases (EC 3.2.1.8). Xylanases have been widely used in many industrial applications and processes, such as in the feed and pulp industries [3, 4]. Xylanases have been used in the production of bioethanol [5]. Xylanases are mainly classified into two glycoside hydrolase (GH) families, named family (GH 10) and family (GH 11). 10 includes endo-β-1,4-xylanases with higher molecular masses (> 30 kDa) and higher thermostabilities than family 11 xylanases.
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