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.
Highlights
Xylanases are increasingly recognized to be important for the deconstruction of lignocellulosic biomass [1,2]
Xylanases catalyze the hydrolysis of hemicelluloses and render cellulose more accessible to enzymatic hydrolysis [3,4,5,6]
That widespread research endeavors have focused on developing thermostable xylanases by rational design or directed evolution
Summary
Xylanases are increasingly recognized to be important for the deconstruction of lignocellulosic biomass [1,2]. The rational design strategy of replacing the N-terminus of mesophilic xylanases with the first 31 residues of the thermophilic xylanase TfxA from Thermomonospora fusca has successfully produced several thermostable hybrid xylanases, such as Stx15 [9], StxAB [10], Btx [11] and ATx [12]. These hybrid xylanases exhibited higher thermostabilities than their corresponding mesophilic parents. A both industrially and biologically intriguing and important question arose: Are mutations in the extreme N-terminus sufficient for conferring hyperthermostability on mesophilic xylanases?
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