Seven N-terminal Residues of a Thermophilic Xylanase Are Sufficient to Confer Hyperthermostability on Its Mesophilic Counterpart

Abstract

Xylanases, and especially thermostable xylanases, are increasingly of interest for the deconstruction of lignocellulosic biomass. In this paper, the termini of a pair of xylanases, mesophilic SoxB and thermophilic TfxA, were studied. Two regions in the N-terminus of TfxA were discovered to be potentially important for the thermostability. By focusing on Region 4, it was demonstrated that only two mutations, N32G and S33P cooperated to improve the thermostability of mesophilic SoxB. By introducing two potential regions into SoxB in combination, the most thermostable mutant, M2-N32G-S33P, was obtained. The M2-N32G-S33P had a melting temperature (Tm) that was 25.6°C higher than the Tm of SoxB. Moreover, M2-N32G-S33P was even three-fold more stable than TfxA and had a Tm value that was 9°C higher than the Tm of TfxA. Thus, for the first time, the mesophilic SoxB “pupil” outperformed its thermophilic TfxA “master” and acquired hyperthermostability simply by introducing seven thermostabilizing residues from the extreme N-terminus of TfxA. This work suggested that mutations in the extreme N-terminus were sufficient for the mesophilic xylanase SoxB to acquire hyperthermostability.

Thermostability improvement of a Talaromyces leycettanus xylanase by rational protein engineering

“…The N-termini of GH11 xylanases play vital roles in thermostability 14 , 16 , 32 . For example, by introducing a disulfide bond, the T m value of TLX was elevated from 66 °C to 74 °C 15 , and the XYNII from Trichoderma reesei had increased thermostability by 11 °C 33 .…”

EcoTILLING in Cochliobolus sativus Isolates Reveals Polymorphisms in the XYL1 and XYL2

“…The ability to break down plant cell walls also has important applications for human endeavours such as in the paper making industry and more recently for the production of biofuels [5]. Although xylanases from eubacteria and archaebacteria have considerably higher temperature optima and stability than those of fungi, the amount of enzyme produced by these bacteria is comparatively lower than that produced by fungi [6][7][8].…”

“…Fungal plant pathogens are considered promising sources of cell wall-degrading enzymes [7][8][9]. The haploid fungus Cochliobolus sativus (anamorph Bipolaris sorokiniana), a necrotrophic cereal pathogen, has been shown to produce cell wall degrading enzymes (CWDEs) including xylanase [10].…”

“…Detecting variations at single nucleotide positions and small nucleotide insertions or deletions are considered to be the most common forms of genetic diversity in natural populations [8,11]. Haploytpe blocks of genetic variation caused by linkage disequilibrium can be detected in different species.…”

Effect of Temperature on Xylanase II from Trichoderma reesei QM 9414: A Calorimetric, Catalytic, and Conformational Study

“…Among the strategies followed to increase the thermal stability of the enzyme, a favourite has been the introduction of disulfide bond by means of site-directed mutagenesis [ 6 , 7 ] as well as the introduction of arginines [ 8 ]. Addition of seven thermophilic derived residues onto a mesophilic xylanase was sufficient to confer thermostability to this engineered variant [ 9 ]. Other attempts of improving xylanase thermostability have been only partially successful since the mutants were thermostable compared with the wild type but less active at low temperatures [ 10 , 11 ].…”