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.

Functionalization of Porous Cellulose with Glyoxyl Groups as a Carrier for Enzyme Immobilization and Stabilization

“…Hence, the inactivation undergone by Xyl during the immobilization on CEL-G1 might rely on the longer incubation time under alkaline conditions demanded by this carrier to achieve high immobilization yields . In fact, free Xyl lost all its activity after 48 h of incubation at pH 10 and 25 °C (Figure S10) as reported for other xylanases inactivated under alkaline conditions. , …”

“…26 In fact, free Xyl lost all its activity after 48 h of incubation at pH 10 and 25 °C (Figure S10) as reported for other xylanases inactivated under alkaline conditions. 27,28 To unveil whether the glyoxyl chemistry was promoting different conformational changes on Xyl immobilized on AG-G and CEL-G1, we recorded the Trp-fluorescence spectra of the immobilized biocatalysts (Figure S11). The normalized spectra showed a significant 5 nm red-shifted maximum emission peak (λ max ) in Xyl immobilized on AG-G regarding the same enzyme immobilized on CEL-G1.…”

Engineering mesophilic GH11 xylanase from Cellulomonas flavigena by rational design of N-terminus substitution

“…The optimum temperature of GH11 xylanase XynJ from Bacillus sp. strain 41M–1 shifted upwards by 5 C–65°C owing to random mutations ( Takita et al, 2021 ) and that of Xyn2 from Trichoderma reesei shifted upwards by 10°C–60°C after site-directed mutagenesis ( He et al, 2019 ). After incubating the CFXyl3 and four hybrid xylanases at pH 7.0 and 70°C for 30–150 min, the stability of the five xylanases was determined ( Figure 4C ).…”

Extremophilic Prokaryotic Endoxylanases: Diversity, Applicability, and Molecular Insights

“…The half-life of mutated xylanase (Q162H/Q162Y, N11D, and N38E) was increased up to 100 min by introducing disulfide bonds ( Turunen et al, 2002 ). Several such cases have been observed where disulfide bonds along with other favorable mutations improved the thermostability up to 5,000-fold ( Xiong et al, 2004 ; He et al, 2019 ). Disulfide bonds and multiple-proline substitutions in acidic xylanase of Aspergillus sulphureus enhanced the thermostability by 22-fold at 60°C ( Yang et al, 2017 ).…”